Heparin-Binding Epidermal Growth Factor-like Growth Factor Binding Proteins

ABSTRACT

Provided herein are antigen binding proteins, e.g., human and/or monoclonal antibodies that have affinity for heparin-binding epidermal growth factor-like growth factor (HB-EGF) and neutralize the biological functions of this growth factor.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/680,191, which is a 371 national stage application ofPCT/EP2008/008233, filed Sep. 26, 2008, which claims priority to andincorporated by reference U.S. provisional application Ser. No.60/975,485, filed Sep. 26, 2007.

BACKGROUND

The human epidermal growth factor receptor (HER) family comprises fourdistinct receptor tyrosine kinases referred to as HER1 (or erbB1), HER2(or erbB2), HER3 (or erbB3), and HER4 (or erbB4). HER1 is also commonlyreferred to as epidermal growth factor receptor (EGFR). With theexception of HER3, these receptors have phospho-acceptor target specificintrinsic protein tyrosine kinase activities. Members of the HER familyare expressed in most epithelial cells as well as in a number ofdifferent tumor cell types. For example, receptors of the HER family areexpressed in tumor cells of epithelial origin, and of mesenchymalorigin. Moreover, HER receptor tyrosine kinases are involved in cellproliferation and angiogenesis, which are associated with diseases suchas cancer. For example, EGFR is frequently over-expressed or aberrantlyactivated in breast cancers, liver cancers, kidney cancers, leukemia,bronchial cancers, pancreatic cancers and gastrointestinal cancers suchas colon, rectal or stomach cancers. High levels of the EGF receptoralso correlate with poor prognosis and response to treatment (Wright etal., 1992, Br. J. Cancer 65:118-121). Thus, disruption of signaltransduction from and to these kinases would have an anti-proliferative,and as such, therapeutic effect upon a number of cancer and tumor celltypes.

The enzymatic activity of receptor tyrosine kinases can be stimulated byover-expression and/or by ligand-mediated dimerization (Heldin, 1995,Cell 80:213-223). Activation of receptor homodimers and heterodimersresults in phosphorylation of tyrosine residues on the receptors, whichin turn phosphorylate tyrosine residues of other molecules, includingintracellular proteins. (Ullrich et al., 1990, Cell 61:203-212). This isfollowed by the activation of intracellular signaling pathways such asthose involving the mitogen-activated protein kinase (MAP kinase)(Dhillon et al., 2007, Oncogene 26: 3279-3290) and thephosphatidylinositol 3-kinase (PI3 kinase). While activation of thesepathways has been shown to increase cell proliferation and inhibitapoptosis, inhibition of signaling mediated by HER family members byeither small molecule inhibitors or monoclonal antibodies has been shownto inhibit cell proliferation and promote apoptosis (Prenzel et al.,2001, Endocr. Relat. Cancer 8: 11-31)

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a22 kDa, O-glycosylated protein (Higahiyama et al., 1992, J Biol Chem267: 6205-6212). In its mature form, HB-EGF binds to and activates theEGF receptor and HER4 (Elenius et al., 1997, EMBO 16:1268-1278). HB-EGFis the key mediator of G-protein coupled receptor (GPCR) induced cellproliferation via a process called triple-membrane passing signaling(TMPS) (Prenzel et al., 1999, Nature 402:884-888, review in Fischer etal. 2003, Biochem. Soc. Trans. 31:1203-1208). It has been shown thatHB-EGF promotes cellular proliferation as well as angiogenesis (Zushi etal., 1997, Int J Cancer 73:917-923; Abramovitch et al., 1998, FEBSletters 425:441-447). HB-EGF also has been demonstrated to play a keyrole in a number of cancers, i.e., it has been linked to the aggressivebehavior of ovarian tumors (Tanaka et al., 2005, Clin. Cancer Res.11:4783-4792). Moreover, HB-EGF is essential for xenograft tumorformation by ovarian cancer cell lines. Over-expression of HB-EGF (wildtype or a secreted form) accelerates tumor formation in SKOV3 and RMG-1cells. Knockdown of endogenous HB-EGF using siRNA, yet, abolished ordelayed tumor formation by SKOV3 and RMG-1 cells. Miyamoto, 2004, CancerRes. 64:5720. As suggested by the above evidence, inhibition of HB-EGFexpression or activity may inhibit tumor formation.

Similarly, HB-EGF is a marker of poor prognosis in some cancers,including human bladder cancers (Thogersen et al., 2001, Cancer Res.61:6227-6233). In vitro studies indicate that human EJ bladder cellsthat were engineered to express HB-EGF (wild type, soluble ornon-cleavable) exhibit an increase in growth, anchorage independentgrowth, and production of VEGF, and enhanced migration. When theseHB-EGF-expressing EJ bladder cells were transplanted into nude mice, anincrease in tumor formation, size and density of blood vessels wasobserved in those tumors. (Ongusaha, 2004, Cancer Res. 64:5283-5290).

SUMMARY

Provided herein are isolated antigen binding proteins that bind HB-EGF.Some of these antigen binding proteins comprise A) one or more lightchain complementary determining regions (CDRLs) consisting of: (i) aCDRL1 selected from SEQ ID NOs:189-217; (ii) a CDRL2 selected from SEQID NOs:218-233; (iii) a CDRL3 selected from SEQ ID NO:234-274; or (iv) aCDRL of (i), (ii) or (iii) that contains one or more amino acidsubstitutions, deletions or insertions of no more than four amino acids.Alternatively, the HB-EGF antigen binding protein may comprise B) one ormore heavy chain complementary determining regions (CDRHs) consistingof: (i) a CDRH1 selected from SEQ ID NO:275-299; (ii) a CDRH2 selectedfrom SEQ ID NO:300-331; (iii) a CDRH3 selected from SEQ ID NO:332-372;or (iv) a CDRH of (i), (ii) or (iii) that contains one or more aminoacid substitutions, deletions or insertions of no more than four aminoacids.

In one embodiment, the isolated antigen binding protein may compriseone, two or more of the aforementioned light chain CDRLs and one, two ormore of the aforementioned heavy chain CDRHs. In one aspect, theisolated antigen binding protein comprises CDRH1, CDRH2, CDRH3, CDRL1,CDRL2 and CDRL3. In another aspect, the isolated antigen binding proteinof A), supra, is selected from the group consisting of: a CDRL1 from SEQID NOs:189-217; a CDRL2 from SEQ ID NOs:218-233; a CDRL3 from SEQ IDNOs:234-274; and a CDRL of the any of the aforementioned (i), (ii) or(ii) that contains one or more amino acid substitutions, deletions orinsertions of no more than two amino acids. In addition, said heavychain CDRH of B), supra, is selected from a CDRH1 from SEQ IDNOs:275-299; a CDRH2 from SEQ ID NOs:300-331; a CDRH3 amino acidsequence from SEQ ID NOs:332-372 and a CDRH of the aforementioned thatcontains one or more amino acid substitutions, deletions or insertionsof no more than two amino acids. Furthermore, the isolated antigenbinding protein may comprise or one or more light chain CDRLs of A),supra; and one or more heavy chain CDRHs of B), supra.

In another embodiment, the antigen binding protein comprises a CDRLselected from the following: a CDRL1 from SEQ ID NOs:189-217; a CDRL2from SEQ ID NOs:218-233; and a CDRL3 from SEQ ID NOs:234-274. Theantigen binding protein may also comprise a CDRH selected from one ofthe following: a CDRH1 from SEQ ID NOs:275-299; a CDRH2 from SEQ IDNOs:300-331; and a CDRH3 selected from SEQ ID NOs:332-372.Alternatively, the isolated antigen binding protein may comprise one ormore light chain CDRLs listed in A), supra, and one or more heavy chainCDRHs of B), supra. In particular, the isolated antigen binding proteinmay comprise a CDRL1 of SEQ ID NOs:189-217, a CDRL2 of SEQ IDNOs:218-233, and a CDRL3 of SEQ ID NOs:234-274 and/or a CDRH1 of SEQ IDNOs:275-299, a CDRH2 of SEQ ID NOs:300-331, and a CDRH3 of SEQ IDNO:332-372.

In one aspect, the isolated antigen binding protein comprises a lightchain variable region (V_(L)) having at least 80%, 90% or 100% sequenceidentity with an amino acid sequence selected from SEQ ID NOs:94-141. Inanother aspect, the isolated antigen binding protein comprises a heavychain variable region (V_(H)) having at least 80%, 90% or 100% sequenceidentity with an amino acid sequence from SEQ ID NOs:142-186.

In another embodiment, the isolated antigen binding protein specificallyrecognizes at least an IHGE containing epitope and/or an EGF-like domainof HB-EGF.

Provided herein, in addition, is an isolated antigen binding proteinthat competes for binding with the isolated antigen binding protein thatbinds HB-EGF, as described above.

Also provided herein is an isolated antigen binding protein which bindsHB-EGF and comprises A) one or more light chain CDRs (CDRLs) from thegroup consisting of: (i) a CDRL1 with at least 80%, or at least 90%sequence identity to SEQ ID NOs:189-217; (ii) a CDRL2 with at least 80%,or at least 90% sequence identity to SEQ ID NOs:218-233; and (iii) aCDRL3 with at least 80%, or at least 90% sequence identity to SEQ IDNOs:234-274. Alternatively, the isolated antigen binding protein whichbinds HB-EGF, comprises B) one or more heavy chain CDRs (CDRHs) from thegroup consisting of (i) a CDRH1 with at least 80%, or at least 90%sequence identity to SEQ ID NOs:275-299; (ii) a CDRH2 with at least 80%,or at least 90% sequence identity to SEQ ID NOs:300-331; and (iii) aCDRH3 with at least 80%, or at least 90% sequence identity to SEQ IDNOs:332-372. The isolated antigen binding protein may also comprise C)one or more light chain CDRLs of A) and one or more heavy chain CDRHs ofB).

In another embodiment, the isolated antigen binding protein binds HB-EGFand comprises: A) a light chain complementary determining region (CDRL)selected from: (i) a CDRL3 selected from the group consisting of SEQ IDNOs:234-274; (ii) a CDRL3 that differs in amino acid sequence from theCDRL3 of (i) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (iii) a CDRL3 amino acid sequenceselected from the following:

X₁QX₂X₃X₄X₅PX₆X₇, (SEQ ID NO: 1046)wherein

-   -   X₁ is I or M,    -   X₂ is A, G or S,    -   X₃ is I or T,    -   X₄ is H or Q,    -   X₅ is F, L or W,    -   X₆ is C, I, H, L or T,    -   X₇ is S or T;

QQX₁X₂X₃X₄X₅IT, (SEQ ID NO: 1047)wherein

-   -   X₁ is I or S,    -   X₂ is F or Y,    -   X₃ is F, I, S or Y,    -   X₄ is A, S or T,    -   X₅ is P or S;

X₁X₂X₃X₄X₅X₆X₇X₈T, (SEQ ID NO: 1048)wherein

-   -   X₁ is L or Q,    -   X₂ is K, N or Q,    -   X₃ is A, H, S or Y,    -   X₄ is H, N or Y,    -   X₅ is N, S or T,    -   X₆ is A, F, I, T, V or Y,    -   X₇ is P or no amino acid,    -   X₈ is F, L or P;

QX₁X₂DX₃LPX₄X₅, (SEQ ID NO: 1049)wherein

-   -   X₁ is H or Q,    -   X₂ is C or Y,    -   X₃ is D, I, N, S or Y,    -   X₄ is F, I or L,    -   X₅ is A, S or T;

QQX₁X₂X₃X₄PX₅X₆X₇, (SEQ ID NO: 1050)wherein

-   -   X₁ is H or Y,    -   X₂ is G or N,    -   X₃ is N or S,    -   X₄ is S or W,    -   X₅ is P or no amino acid,    -   X₆ is R or W,    -   X₇ is S or T; or

X₁QYX₂X₃X₄X₅X₆X₇F, (SEQ ID NO: 1051)wherein

-   -   X₁ is H or Q,    -   X₂ is F or Y,    -   X₃ is G, I or S,    -   X₄ is F, I or T,    -   X₅ is M, P, S or T,    -   X₆ is F, L, R or W,    -   X₇ is S or T.

The isolated antigen binding protein may also comprise B) a heavy chaincomplementary determining region (CDRH) selected from the groupconsisting of: (i) a CDRH3 selected from the group consisting of SEQ IDNOs:332-372; (ii) a CDRH3 that differs in amino acid sequence from theCDRH3 of (i) by an amino acid addition, deletion or substitution of notmore than two amino acids; and iii) a CDRH3 amino acid sequence selectedfrom the following:

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁DX₁₂, (SEQ ID NO: 1065)wherein

-   -   X₁ is E or S,    -   X₂ is D, G or no amino acid,    -   X₃ is D, N or no amino acid,    -   X₄ is G or no amino acid,    -   X₅ is G or no amino acid,    -   X₆ is W, Y or no amino acid,    -   X₇ is I, N or Y,    -   X₈ is A or Y,    -   X₉ is G, V or Y,    -   X₁₀ is A, F or G,    -   X₁₁ is F, L or M,    -   X₁₂ is V or Y;

(SEQ ID NO: 1066) QX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁YX₁₂X₁₃X₁₄DX₁₅,wherein

-   -   X₁ is G or no amino acid,    -   X₂ is K, L or Y,    -   X₃ is A, G or S,    -   X₄ is S, V or Y,    -   X₅ is A or G,    -   X₆ is G or no amino acid,    -   X₇ is T or no amino acid,    -   X₈ is S or no amino acid,    -   X₉ is Y or no amino acid,    -   X₁₀ is W or Y,    -   X₁₁ is G, S or Y,    -   X₁₂ is F or Y,    -   X₁₃ is G or no amino acid,    -   X₁₄ is M or no amino acid,    -   X₁₅ is V or Y;

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄, (SEQ ID NO: 1067)wherein

-   -   X₁ is D, G, L, S or no amino acid,    -   X₂ is G, H, W, Y or no amino acid,    -   X₃ is A, F, W, Y or no amino acid,    -   X₄ is D, G, Q, T or no amino acid,    -   X₅ is G, I, Q, S or no amino acid,    -   X₆ is A, D, N, Q, S or no amino acid,    -   X₇ is G, Y or no amino acid,    -   X₈ is D, Y or no amino acid,    -   X₉ is Y or no amino acid,    -   X₁₀ is A, E, N or Y,    -   X₁₁ is G, P, T, V or Y,    -   X₁₂ is F or I,    -   X₁₃ is D or Q,    -   X₁₄ is C, H, V or Y;

(SEQ ID NO: 1068) X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇DX₁₈,wherein

-   -   X₁ is E, D or no amino acid,    -   X₂ is G, R or no amino acid,    -   X₃ is I, V, Y or no amino acid,    -   X₄ is A, G, L or N,    -   X₅ is A, G, V or W,    -   X₆ is A, N, R or T,    -   X₇ is G, N, P or no amino acid,    -   X₈ is G, T or no amino acid,    -   X₉ is A or no amino acid,    -   X₁₀ is D, E or no amino acid,    -   X₁₁ is S, Y or no amino acid,    -   X₁₂ is G, Y or no amino acid,    -   X₁₃ is N, Y or no amino acid,    -   X₁₄ is Y or no amino acid,    -   X₁₅ is D, Y or no amino acid,    -   X₁₆ is A, G or no amino acid,    -   X₁₇ is F or M,    -   X₁₈ is I, V or Y;

(SEQ ID NO: 1069) X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃,wherein

-   -   X₁ is A, D, G, S or T,    -   X₂ is A, E, G, L, N, R, Y or no amino acid,    -   X₃ is A, G, L, N, R, T, Y or no amino acid,    -   X₄ is D, G, R, S, V, Y or no amino acid,    -   X₅ is A, G, I, S, V, Y or no amino acid,    -   X₆ is F, G, L, R, V or no amino acid,    -   X₇ is L, T, Y or no amino acid,    -   X₈ is Y or no amino acid,    -   X₉ is Y or no amino acid,    -   X₁₀ is D or no amino acid,    -   X₁₁ is S or no amino acid,    -   X₁₂ is S or no amino acid,    -   X₁₃ is G or no amino acid,    -   X₁₄ is D, L, M, S, Y or no amino acid,    -   X₁₅ is H, I, P, V, W or no amino acid,    -   X₁₆ is F, G, L, R, S, Y or no amino acid,    -   X₁₇ is D, F, V, W, Y or no amino acid,    -   X₁₈ is C, F, L, P, S or Y,    -   X₁₉ is D, F, G or Y,    -   X₂₀ is A, C, G, P, R, V or Y,    -   X₂₁ is F, L, M, S or no amino acid,    -   X₂₂ is A, D or no amino acid,    -   X₂₃ is I, L, V, Y or no amino acid;

X₁YSSGWX₂X₃YGX₄X₅DX₆, (SEQ ID NO: 1070)wherein

-   -   X₁ is M or V,    -   X₂ is S or no amino acid,    -   X₃ is F or no amino acid,    -   X₄ is V or no amino acid,    -   X₅ is F or M,    -   X₆ is V or Y; or

RX₁X₂X₃PFX₄Y, (SEQ ID NO: 1071)wherein

-   -   X₁ is G, H, L, N or R,    -   X₂ is E, T or W,    -   X₃ is L, N, T or V,    -   X₄ is D or E.

In another aspect, the isolated antigen binding protein may furthercomprise A) a CDRL selected from: (i) a CDRL1 selected from SEQ IDNOs:189-217; (ii) a CDRL1 that differs in amino acid sequence from theCDRH1 of (i) by an amino acid addition, deletion or substitution of notmore than two amino acids; or (iii) a CDRL1 amino acid sequence from thefollowing:

X₁SSQSLX₂X₃SDGX₄TYLX₅, (SEQ ID NO: 1035)wherein

-   -   X₁ is K or R,    -   X₂ is L or V,    -   X₃ is H or Y,    -   X₄ is K or N,    -   X₅ is N, S or Y;

RASQX₁ISX₂YLN, (SEQ ID NO: 1036)wherein

-   -   X₁ is R, S or T,    -   X₂ is R or S;

RASQX₁IX₂X₃X₄LX₅, (SEQ ID NO: 1037)wherein

-   -   X₁ is D, G, S or T,    -   X₂ is A, R or S,    -   X₃ is H, I, N, R, S or T,    -   X₄ is D, W or Y,    -   X₅ is A, G or N;

QASQDIX₁X₂X₃LN, (SEQ ID NO: 1038)wherein

-   -   X₁ is S or T,    -   X₂ is D or N,    -   X₃ is S or Y;

RASQX₁VX₂X₃X₄X₅LA, (SEQ ID NO: 1039)wherein

-   -   X₁ is S or T,    -   X₂ is I or S,    -   X₃ is R or S,    -   X₄ is S, N or no amino acid,    -   X₅ is Y or no amino acid; or

KSSQX₁X₂LX₃X₄SNNKNYLX₅, (SEQ ID NO: 1040)wherein

-   -   X₁ is N or S,    -   X₂ is I or V,    -   X₃ is D or Y,    -   X₄ is N, R or 5,    -   X₅ is A or V;

(iv) a CDRL2 from the group consisting of SEQ ID NOs:218-233; (v) aCDRL2 that differs in amino acid sequence from the CDRL2 of (iv) by anamino acid addition, deletion or substitution of not more than two aminoacids; or (vi) a CDRL2 amino acid sequence from the following:

X₁X₂SNX₃X₄S, (SEQ ID NO: 1041)wherein

-   -   X₁ is E or K,    -   X₂ is I or V,    -   X₃ is R or W,    -   X₄ is D or F;

X₁X₂SX₃LQS, (SEQ ID NO: 1042)wherein

-   -   X₁ is A or T,    -   X₂ is A, E or V,    -   X₃ is S or T;

X₁ASX₂LQS, (SEQ ID NO: 1043)wherein

-   -   X₁ is A or V,    -   X₂ is S or T;

DASX₁LET, (SEQ ID NO: 1044)wherein

-   -   X₁ is I or N;

GASSRAT; (SEQ ID NO: 223) or WASX₁RES, (SEQ ID NO: 1045)wherein

-   -   X₁ is A or T.

The isolated antigen binding proteins may further comprise B) a CDRHfrom the group consisting of: (i) a CDRH1 from the group consisting ofSEQ ID NOs:275-299; (ii) a CDRH1 that differs in amino acid sequencefrom the CDRH1 of (i) by an amino acid addition, deletion orsubstitution of not more than two amino acids; (iii) a CDRH1 amino acidsequence selected from:

GYTX₁TX₂X₃X₄X₅X₆, (SEQ ID NO: 1052)wherein

-   -   X₁ is F or L,    -   X₂ is E, G or S,    -   X₃ is H, L or Y,    -   X₄ is G, S or Y,    -   X₅ is I or M,    -   X₆ is H or S;

GYX₁FTSYWIG, (SEQ ID NO: 1053)wherein

-   -   X₁ is R or S;

GFTFX₁SX₂X₃MH, (SEQ ID NO: 1054)wherein

-   -   X₁ is R or S,    -   X₂ is H or Y,    -   X₃ is D or G;

GFX₁FSX₂YX₃MX₄, (SEQ ID NO: 1055)wherein

-   -   X₁ is P or T,    -   X₂ is A, R or S,    -   X₃ is A or S,    -   X₄ is N or S;

GX₁SX₂SX₃X₄X₅X₆X₇WX₈, (SEQ ID NO: 1056)wherein

-   -   X₁ is D or G,    -   X₂ is F, I or V,    -   X₃ is R, S or no amino acid,    -   X₄ is G, Y or no amino acid,    -   X₅ is D, G, S or no amino acid,    -   X₆ is A, S or Y,    -   X₇ is A or Y,    -   X₈ is N or S;

GFSLSNARMGVS; (SEQ ID NO: 279) or GFSLX₁TGGVGVG, (SEQ ID NO: 1057)wherein

-   -   X₁ is S or N;

(iv) a CDRH2 selected from the group consisting of SEQ ID NOs:300-331;(v) a CDRH2 that differs in amino acid sequence from the CDRH2 of (iv)by an amino acid addition, deletion or substitution of not more than twoamino acids; or (vi) a CDRH2 amino acid sequence from the following:

X₁X₂X₃X₄X₅X₆GX₇TX₈X₉X₁₀QKX₁₁X₁₂, (SEQ ID NO: 1058)wherein

-   -   X₁ is S or W,    -   X₂ is F or I,    -   X₃ is D, N or S,    -   X₄ is A or P,    -   X₅ is E, N or S,    -   X₆ is D, N or S,    -   X₇ is E, G or N,    -   X₈ is I or N,    -   X₉ is C, H or Y,    -   X₁₀ is A or T,    -   X₁₁ is F or L,    -   X₁₂ is D or G;

IIYPX₁DSDX₂RYSPSFQG, (SEQ ID NO: 1059)wherein

-   -   X₁ is D or G,    -   X₂ is A, I or T;

X₁IX₂X₃DGSX₄X₅X₆YX₇DSVX₈G, (SEQ ID NO: 1060)wherein

-   -   X₁ is F or V,    -   X₂ is S or W,    -   X₃ is D, S or Y,    -   X₄ is I, N or T,    -   X₅ is K or Q,    -   X₆ is N, R or Y,    -   X₇ is A, T or V,    -   X₈ is K or R;

X₁ISX₂SX₃X₄X₅X₆YYADSVKG, (SEQ ID NO: 1061)wherein

-   -   X₁ is A, H or Y,    -   X₂ is G, R or S,    -   X₃ is G or S,    -   X₄ is G, R or S,    -   X₅ is S, T or Y,    -   X₆ is I or T;

(SEQ ID NO: 1062) X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁YX₁₂X₁₃SX₁₄KS,wherein

-   -   X₁ is E, R or Y,    -   X₂ is I or T,    -   X₃ is H, N or Y,    -   X₄ is C, H, S, T or Y,    -   X₅ is S or R,    -   X₆ is G or S,    -   X₇ is G, K, S or T,    -   X₈ is T or W,    -   X₉ is N or Y,    -   X₁₀ is N or no amino acid,    -   X₁₁ is D or no amino acid,    -   X₁₂ is A or N,    -   X₁₃ is P or V,    -   X₁₄ is L or V;

X₁IFSNDEKSYSTSLKS, (SEQ ID NO: 1063)wherein

-   -   X₁ is H or LI; or

LIYWNX₁X₂KRYSPSLX₃S, (SEQ ID NO: 1064)wherein

-   -   X₁ is D or V,    -   X₂ is D or E,    -   X₃ is K or R.

In yet another embodiment, the isolated antigen binding proteindescribed hereinabove comprises the first amino acid sequence and thesecond amino acid sequence, both sequences of which are covalentlybonded to each other. The first amino acid sequence also comprises CDRL3of SEQ ID NOs:234-274, CDRL2 of SEQ ID NOs:218-233, and CDRL1 of SEQ IDNOs:189-217, and the second amino acid sequence comprises said CDRH3 ofSEQ ID NOs:332-372, CDRH2 of SEQ ID NOs:300-331, and CDRH1 of SEQ IDNOs:275-299.

In one aspect, the isolated antigen binding proteins can be a monoclonalantibody, a polyclonal antibody, a recombinant antibody, a humanantibody, a humanized antibody, a chimeric antibody, a multispecificantibody, or an antibody fragment thereof. The antibody fragment may bea Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment, a Fv fragment, adiabody, or a single chain antibody molecule. In one embodiment, theisolated antigen binding protein of the present invention is a humanantibody. In another embodiment, the isolated antigen binding protein isa monoclonal antibody.

The isolated antigen binding proteins as described herein, may be of anyof the following types: IgG1-, IgG2-IgG3- or IgG4-type. In oneembodiment, the antigen binding protein is of the IgG2- or IgG4-type.Furthermore, the antigen binding protein may be coupled to a labelinggroup. These labeling groups may be, for example, a radioisotope,radionuclide, a fluorescent group, an enzymatic group, achemiluminescent group, a biotinyl group, or a predetermined polypeptidegroup.

In another embodiment, the isolated antigen binding protein is coupledto an effector group such as, for example, a radioisotope, aradionuclide, a toxin, a therapeutic group, or a chemotherapeutic group.The chemotherapeutic groups may be, for example, calicheamicin,auristatin-PE, geldanamycin, maytanasine, or derivatives thereof.

In yet another embodiment, the isolated antigen binding protein competesfor binding to human HB-EGF with a antigen binding protein as describedand claimed herein. This competing antigen binding protein may be, forexample, a monoclonal antibody, a polyclonal antibody, a recombinantantibody, a human antibody, a humanized antibody, a chimeric antibody, amultispecific antibody, or an antibody fragment thereof. The antibodyfragment may be, for example, a Fab fragment, a Fab′ fragment, a F(ab)₂fragment, a Fv fragment, a diabody, or a single chain antibody molecule.In one embodiment, the isolated binding protein is a human antibody. Inanother embodiment, the isolated antigen binding protein is a monoclonalantibody. In another embodiment, this isolated antigen binding proteinis of the IgG1-, IgG2-IgG3- or IgG4-type. In one embodiment, the antigenbinding protein is of the IgG2- or IgG4-type. In another embodiment, theantigen binding proteins as described herein can be coupled to alabeling group. Examples of labeling groups are: a radioisotope,radionuclide, a fluorescent group, an enzymatic group, achemiluminescent group, a biotinyl group, or a predetermined polypeptidegroup. In another embodiment, the isolated antigen binding protein iscoupled to an effector group such as, for example, a radioisotope, aradionuclide, a toxin, a therapeutic group, or a chemotherapeutic group.Examples of the therapeutic or chemotherapeutic groups include, forexample, calicheamicin, auristatin-PE, geldanamycin, maytanasine, orderivatives thereof.

In one aspect, an isolated antigen binding protein is provided thatreduces, at least partially, HB-EGF-mediated signal transduction.

Also presented herein is a nucleic acid molecule encoding the isolatedantigen binding protein previously described, wherein the nucleic acidmolecule is operably linked to a control sequence. In one aspect, avector comprising the aforementioned nucleic acid molecule is provided.In another aspect, a host cell is provided that comprises theaforementioned nucleic acid molecule and/or vector.

In one embodiment, a method for making the antigen binding protein isprovided that includes the step of preparing said antigen bindingprotein from a host cell that secretes said antigen binding protein.

In yet another embodiment, a pharmaceutical composition is providedcomprising at least one of the aforementioned antigen binding proteinsof the present invention and a pharmaceutically acceptable carrier,diluent or adjuvant. In one embodiment, the pharmaceutical compositionmay comprise an additional active agent, such as an anti-neoplasticagent. The anti-neoplastic agent may be, for example, an anti-tumorantibody. Examples of an anti-tumor antibody may be, for example,antibodies directed against receptor tyrosine kinase or EGFR.

In one aspect, the pharmaceutical composition is used for diagnosis,prevention or treatment of a hyperproliferative disease. In a furtheraspect, the hyperproliferative disease is associated with HB-EGFexpression. In another aspect, the hyperproliferative disease isassociated with or accompanied by a disturbed, (e.g. pathologicallyenhanced), growth factor receptor activation, wherein saidpathologically enhanced growth factor receptor activation is associatedwith or caused by a pathological increase in the activity of a G proteinand/or a G protein coupled receptor.

In one embodiment, the pharmaceutical composition comprises at least oneantigen binding protein and pharmaceutically acceptable carrier,diluents and/or adjuvants for the diagnosis, prevention or treatment ofcancer, such as, for example, breast cancer, gastrointestinal cancer,pancreas cancer, prostate cancer, ovarian cancer, stomach cancer,endometrial cancer, salivary gland cancer, lung cancer, kidney cancer,colon cancer, colorectal cancer, thyroid cancer, bladder cancer, glioma,melanoma, other HB-EGF expressing or overexpressing cancers, andformation of tumor metastases.

In another embodiment, antigen binding proteins as described herein areused for the manufacture of a pharmaceutical composition for thediagnosis, prevention or treatment of a hyperproliferative disease. In afurther embodiment, the hyperproliferative disease is associated withHB-EGF expression.

One embodiment describes a method for diagnosing a condition associatedwith the expression of HB-EGF, the method comprising the step ofcontacting a sample an isolated antigen binding proteins as describedherein, and determining the presence of HB-EGF in said sample. In afurther embodiment, the condition is a hyperproliferative diseaseassociated with HB-EGF expression.

Another aspect describes a method for preventing or treating a conditionassociated with the expression of HB-EGF in a patient, comprisingadministering to a patient in need thereof an effective amount of aantigen binding protein as described herein. In a further aspect, thecondition is a hyperproliferative disease associated with HB-EGFexpression. In yet another aspect, the patient is a mammalian patient.

In one embodiment, a kit is provided that comprises a antigen bindingprotein, a nucleic acid molecule, or a vector as described above. In afurther embodiment, the kit comprises at least one further active agent,wherein the further active agent is an anti-neoplastic agent.

These and other aspects of the invention will be described in greaterdetail herein. Each of the aspects of the invention can encompassvarious embodiment of the present invention. It is therefore anticipatedthat each of the embodiments of the invention involving one element orcombinations of elements can be included in each aspect of theinvention. Other features, objects, and advantages of the presentinvention are apparent in the detailed description that follows.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1P depict various light chain variable regions of the antigenbinding proteins. The CDR1, CDR2 and CDR3 regions are indicated inboxes.

FIGS. 2A-2O depict various heavy chain variable regions of the antigenbinding proteins. The CDR1, CDR2 and CDR3 regions are indicated inboxes.

FIGS. 3A-3K depict the amino acid sequences of various light chains ofthe antigen binding proteins.

FIGS. 4A-4O depict the amino acid sequences of various heavy chains ofthe antigen binding proteins.

FIG. 5A depicts the amino acid sequence of an exemplary light chainconstant region of the antigen binding proteins.

FIG. 5B depicts the amino acid sequence of an exemplary heavy chainconstant region of the antigen binding proteins.

FIGS. 6A-6F depict the amino acid sequences for various CDR regions ofthe light chain variable regions of the antigen binding proteins.

FIGS. 7A-7E depict the amino acid sequences for various CDR regions ofthe heavy chain variable regions of the antigen binding proteins.

FIGS. 8A-8H depict the amino acid sequences for various FR regions ofthe light chain variable regions of the antigen binding proteins.

FIGS. 9A-9F depict the amino acid sequences for various FR regions ofthe heavy chain variable regions of the antigen binding proteins.

FIGS. 10A and 10B depict an alignment of the amino acid sequences of thelight chain variable sequences of the antigen binding proteins. TheCDR1, CDR2 and CDR3 regions are shown in boxes.

FIGS. 11A and 11B depict an alignment of the amino acid sequences of theheavy chain variable sequences of the antigen binding proteins. TheCDR1, CDR2 and CDR3 regions are shown in boxes.

FIG. 12A depicts a cladogram showing the relatedness of the light chainvariable regions of the antigen binding proteins.

FIG. 12B depicts a cladogram showing the relatedness of the light chainCDRL3 regions of the antigen binding proteins.

FIG. 12C depicts a cladogram showing the relatedness of the heavy chainvariable regions of the antigen binding proteins.

FIGS. 13A-13V depict the nucleotide sequences of various light chainvariable regions of the antigen binding proteins.

FIGS. 14A-14AC depict the nucleotide sequences of various heavy chainvariable regions of the antigen binding proteins.

FIGS. 15A-15M depict the nucleotide sequences of the various lightchains of the antigen binding proteins.

FIGS. 16A-16L depict the nucleotide sequences of the various heavychains of the antigen binding proteins.

FIG. 17A depicts the nucleotide sequence of the light chain constantregion of the antigen binding proteins.

FIG. 17B depicts the nucleotide sequence of the heavy chain constantregion of the antigen binding proteins.

FIGS. 18A-18F depict the nucleotide sequences for various CDR regions ofthe light chain variable regions of the antigen binding proteins.

FIGS. 19A-19G depict the nucleotide sequences for various CDR regions ofthe heavy chain variable regions of the antigen binding proteins.

FIGS. 20A-20K depict the nucleotide sequences for various FR regions ofthe light chain variable regions of the antigen binding proteins.

FIGS. 21A-21K depict the nucleotide sequences for various FR regions ofthe heavy chain variable regions of the antigen binding proteins.

FIG. 22A graphically illustrates the degree to which differentanti-HB-EGF IgG2 antibody preparations provided herein inhibitHB-EGF-induced epidermal growth factor receptor (EGFR) tyrosinephosphorylation. The results for preparations of antibodies U2-1 toU2-68 are provided. As illustrated, monoclonal antibody preparationsU2-18, U2-24, U2-19 and U2-42 strongly inhibit EGFR tyrosinephosphorylation.

FIG. 22B graphically illustrates the degree to which differentanti-HB-EGF IgG4 antibody preparations provided herein inhibitHB-EGF-induced epidermal growth factor receptor (EGFR) tyrosinephosphorylation. The results for preparations of antibodies U2-2 toU2-66 are provided. As illustrated, monoclonal antibody preparationsU2-39, U2-34, U2-45 and U2-6 strongly inhibit EGFR tyrosinephosphorylation.

FIG. 23 illustrates that the antibodies inhibit lysophosphatidic acid(LPA)-induced EGFR tyrosine phosphorylation in COS-7 cells. LPA is aGPCR ligand that activates the TMPS pathway, resulting in release ofHB-EGF with consequent EGFR tyrosine phosphorylation. COS-7 cells werepretreated with antibodies as indicated and stimulated with LPA, thencell lysates were prepared and lysate proteins were separated bypolyacrylamide gel electrophoresis. After preparation of the blot, ananti-phosphotyrosine antibody was used to detect phosphorylated EGFR. Asa control, total EGFR was detected as shown at the bottom, using a WBanti-EGFR antibody. As illustrated, anti-HB-EGF antibody preparationsU2-24, U2-19 and U2-42 strongly inhibit LPA-induced EGFRphosphorylation.

FIG. 24 graphically illustrates dose-dependent inhibition ofHB-EGF-induced EGF receptor tyrosine phosphorylation by variousantibodies provided herein. Different concentrations of the candidateU2-39, U2-42 and U2-45 antibody preparations were preincubated withHB-EGF prior to stimulation of SCC9 squamous cancer cells and the amountof EGFR tyrosine phosphorylation was detected. As shown, antibody U2-42and U2-39 achieved up to 111% inhibition. IC50 values determined for theantibodies were 0.167 nM (U2-39), 1 nM (U2-42) and 2 nM (U2-45),respectively.

FIG. 25 graphically illustrates dose-dependent inhibition ofthrombin-induced EGFR phosphorylation via TMPS in MDA-MB231 cells byanti-HB-EGF antibody preparations. MDA-MB231 cells were incubated withcandidate U2-42, U2-39 and U2-45 antibody preparations in the presenceof thrombin and the amount of EGFR tyrosine phosphorylation was detectedusing a procedure described in Example 6. As shown, antibodies U2-42 andU2-39 achieving 100% inhibition.

FIG. 26 illustrates dose-dependent inhibition of LPA-induced EGFRtyrosine phosphorylation via TMPS in PPC-1 cells by anti-HB-EGF antibodypreparations. PPC-1 cells were incubated with candidate U2-42, U2-39 andU2-45 antibody preparations and the amount of EGFR tyrosinephosphorylation following LPA stimulation was detected using a proceduredescribed in Example 4. As shown, antibodies U2-42, U2-39 and U2-45achieved 100% inhibition.

FIG. 27 illustrates that anti-HB-EGF antibody preparations inhibited byup to 100% the induction of MDA-MB231 breast cancer cell migration bysphingosine-1-phosphate. Candidate anti-HB-EGF antibody preparationsU2-42, U2-39 and U2-45 were tested for cell migration inhibition usingof collagen I-coated transwells (BD Falcon, 8 μm pores). As shown,anti-HB-EGF antibody preparation U2-42 inhibitedsphingosine-1-phosphate-induced MDA-MB231 cell migration by about 70%while the U2-39 and U2-45 anti-HB-EGF antibody preparations inhibitedMDA-MB231 cell migration by about 100%. Thus, the anti-HB-EGF antibodiesprovided herein strongly inhibit MDA-MB231 cell migration.

FIG. 28 graphically illustrates that HB-EGF-induced migration of MCF-7breast cancer cells is inhibited by three anti-HB-EGF antibodypreparations (the U2-42, the U2-39 and the U2-45 monoclonal antibodypreparations).

FIG. 29 illustrates the dose-dependent inhibition of HB-EGF-inducedtyrosine phosphorylation of HER4 by anti-HB-EGF antibody preparations.U2-42.1 or U2-39.1 anti-HB-EGF antibody preparations were incubated withHB-EGF prior to stimulation and detection of HER4 tyrosinephosphorylation. As shown, 100% inhibition of HER4 tyrosinephosphorylation was observed. Note that the amount of antibody shown onthe x-axis decreases logarithmically.

FIG. 30A shows that monoclonal antibody preparations cross-react withHB-EGF from cynomolgus monkeys as assessed by flow cytometry (FACS)using HEK-293 cells transfected with a DNA vector expressing cynomolgusHB-EGF. As shown, very low X-mean values (1-2) are observed for HEK-293control cells that were transfected with an empty vector control. Incontrast, X-mean values of 250 or more were observed when HEK-293 cellswere transfected with an expression cassette encoding cynomolgus monkeyHB-EGF.

FIG. 30B shows that monoclonal antibody U2-45 preparation cross-reactswith HB-EGF from mouse as assessed by flow cytometry (FACS) usingHEK-293 cells transfected with a DNA vector expressing mouse HB-EGF.X-mean values of 33.7 were observed when HEK-293 cells were transfectedwith an expression cassette encoding mouse HB-EGF.

FIG. 30C shows the degree of cross-reactivity of HB-EGF antibodies withamphiregulin.

FIG. 31 shows that HB-EGF is expressed on human vascular endothelialcells (HUVECs), as detected by FACS analysis.

FIGS. 32A-32B show that while HB-EGF stimulates HUVEC cellularproliferation, anti-HB-EGF antibody preparations inhibited basalproliferation by about 8% to 14%. HB-EGF stimulates HUVEC cellularproliferation by about 38% (FIG. 32A). However, upon addition ofanti-HB-EGF antibody preparations U2-42, U2-39 or U2-45, basal cellularproliferation is inhibited by about 8% to 14% (FIG. 32B).

FIGS. 33A-33L illustrate that anti-HB-EGF antibodies accelerate HUVECtube regression. HUVEC tube formation is a model system for endothelialcell angiogenesis. FIGS. 33A-33C provide control assays that wereperformed without anti-HB-EGF antibodies. As shown, HUVEC cells join toform many circular structures or “tubes.” FIGS. 33D-33F illustrate theeffects of adding the anti-HB-EGF U2-39 antibody preparation upon tubeformation. FIGS. 33G-33I illustrate the effects of adding theanti-HB-EGF U2-42 antibody preparation upon tube formation. FIGS.33J-33L illustrate the effects of adding the anti-HB-EGF U2-45 antibodypreparation upon tube formation. As shown, fewer HUVEC tubes are visibleand the network is diminished when the U2-42, U2-39 and U2-45anti-HB-EGF antibody preparations are present.

FIG. 33M graphically illustrates a quantitative evaluation of HUVEC tubeformation after adding the anti-HB-EGF antibody preparations providedherein, supporting the utility of HB-EGF antibodies for inhibitingangiogenesis. The number of tubes or closed cell structures permicroscopic field is plotted for the U2-42, U2-39 or U2-45 anti-HB-EGFantibody preparations. As shown, while approximately 10 HUVEC tubes werevisible per field when no anti-HB-EGF antibodies were present, onlyabout 4 HUVEC tubes were observed per microscopic field when the U2-39anti-HB-EGF antibody preparation was added. In the presence of the U2-42anti-HB-EGF antibody preparation only about 1 HUVEC tube was observed.When the U2-45 anti-HB-EGF antibody preparation was present only 6 HUVECtubes were observed.

FIG. 34A illustrates that anti-HB-EGF antibodies inhibitHB-EGF-stimulated colony formation of OVCAR-8 ovarian cancer cells. Asshown, HB-EGF stimulated OVCAR-8 cells to form a significantly largermean colony size than control OVCAR-8 cells cultured without HB-EGF.However, when OVCAR-8 cells were cultured with anti-HB-EGF U2-39antibodies in the presence of HB-EGF, mean colony size was reduced tothe baseline size observed for control cells without HB-EGF treatment.

FIG. 34B illustrates that anti-HB-EGF antibodies inhibitHB-EGF-stimulated colony formation of BM1604 prostate cancer cells insoft agar. As shown, HB-EGF stimulated BM1604 cells to form a largernumber of colonies per well than control BM1604 cells cultured withoutHB-EGF. However, when BM1604 cells were cultured with anti-HB-EGF U2-39antibodies in the presence of HB-EGF, mean colony size was reduced to asize similar to that observed for control cells without HB-EGFtreatment. Anti-HB-EGF U2-45 and U2-42 antibodies partially inhibitedcolony formation, while, in this assay, the U2-39 anti-HB-EGF antibodycompletely inhibited colony formation.

FIG. 34C illustrates that anti-HB-EGF antibodies inhibitHB-EGF-stimulated colony formation of NCI-H226 lung carcinoma cells. Asshown, HB-EGF stimulated NCI-H226 cells to form a significantly largermean colony size than control NCI-H226 cells cultured without HB-EGF.However, when NCI-H226 cells were cultured with anti-HB-EGF U2-39antibodies in the presence of HB-EGF, mean colony size was reduced tothe baseline size observed for control cells without HB-EGF treatment.

FIG. 34D illustrates that anti-HB-EGF antibodies inhibit basal colonyformation of SkOV-3 HB-EGF clone 71 cells, derived from SkOV-3 ovariancancer cells transfected with an HB-EGF expression vector to causeconstitutive over-expression of HB-EGF. As shown, control SkOV-3 HB-EGFclone 71 cells formed large numbers of colonies. However, when SkOV-3HB-EGF cl. 71 cells were cultured with either anti-HB-EGF U2-42 or U2-39antibodies, the number of colonies was dramatically reduced.

FIG. 34E illustrates that anti-HB-EGF antibodies inhibit basal colonyformation of SkOV-3 HB-EGFclone 74 cells, derived from SkOV-3 ovariancancer cells transfected with an HB-EGF expression vector to causeconstitutive over-expression of HB-EGF. As shown, control SkOV-3 HB-EGFclone 74 cells formed large numbers of colonies. However, when SkOV-3HB-EGF clone 74 cells were cultured with anti-HB-EGF U2-39 antibodies,the number of colonies was dramatically reduced.

FIG. 34F illustrates that anti-HB-EGF antibodies inhibit basal colonyformation of BxPC3 pancreatic adenocarcinoma cells grown in soft agar.As shown, control BxPC3 cells formed large numbers of colonies. However,when BxPC3 cells were cultured with either anti-HB-EGF U2-42 or U2-39antibodies in the presence of HB-EGF, the number of colonies wasdramatically reduced.

FIG. 35 illustrates that anti-HB-EGF antibodies inhibit basal colonyformation of EFO-27 HB-EGF clone 58 ovarian cancer cells overexpressingHB-EGF grown in soft agar. As shown, control cells formed large numbersof colonies. However, when EFO-27 HB-EGF cl. 58 cells were cultured witheither anti-HB-EGF U2-42, U2-39 or U2-45 antibodies the number ofcolonies was dramatically reduced. Moreover, combination therapy ofanti-HB-EGF antibodies with the anti-EGFR antibody Erbitux completelyinhibited the colony formation.

FIG. 36 shows that anti-HB-EGF antibodies inhibit HB-EGF inducedangiogenesis in vivo. Angiogenic network formation in a mouse matrigelplug assay could be blocked in a dose-dependent manner by antibodiesU2-42, U2-39 and U2-45.

FIG. 37 illustrates inhibition of the growth of established BxPC3 tumorsin mouse xenograft models by antibodies U2-42 and U2-39.

FIGS. 38A-38C illustrate inhibition of the growth of established EFO-27HB-EGF clone 58 tumors in mouse xenograft models by antibodies U2-42,U2-39 and U2-45 (FIG. 38A). As shown in FIG. 38B, efficacy of inhibitionof the xenograft tumor growth by antibodies U2-42 and U2-39 was shown tobe dose dependent. Moreover, combination treatment with the anti-EGFRantibody Erbitux leads to complete regression of tumor growth and showsthe potent synergistic activity of the anti-HB-EGF antibodies as agentsfor combination therapy (FIG. 38C).

FIGS. 39A-39B illustrate the use of the human anti-HB-EGF antibodies fordetection of HB-EGF in human tissue by immunohistochemistry (FIG. 39A)and by ELISA (FIG. 39B).

FIG. 40A illustrates a scratch assay indicating the inhibition ofHB-EGF-induced migration of CLS354 epithelial squamous carcinoma cells(mouth).

FIG. 40B illustrates a transmigration assay indicating the inhibition ofHB-EGF-induced migration of Detroit 562 epithelial carcinoma cells(pharynx).

FIGS. 41A and B illustrate a spheroid-based cellular angiogenesis assayindicating the inhibition of VEGF-stimulated endothelial cell sprouting.

FIG. 42 illustrates immunohistochemistry (IHC) analysis of human tumorxenograft samples indicating the inhibition of CD31 staining of tumor invivo.

FIGS. 43A and 43B illustrate in vivo ovarian tumor xenograft modelindicating combination treatment of U2-39 with Cisplatin and Avastin.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present application are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001) and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), which are incorporated herein byreference. Enzymatic reactions and purification techniques are performedaccording to manufacturers specifications, as commonly accomplished inthe art or as described herein. The terminology used in connection with,and the laboratory procedures and techniques of, analytical chemistry,synthetic organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein are those well known and commonly used in the art.Standard techniques can be used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the disclosed, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean ±1%.

A. General Overview

Antigen binding proteins that bind HB-EGF protein, in particular humanHB-EGF (hHB-EGF) protein are provided herein. The antigen bindingproteins provided are polypeptides into which one or more complementarydetermining regions (CDRs), as described herein, are embedded and/orjoined. In some antigen binding proteins, the CDRs are embedded into a“framework” region, which orients the CDR(s) such that the properantigen binding properties of the CDR(s) is achieved. In general,antigen binding proteins that are provided can interfere with, block,reduce or modulate the interaction between HB-EGF and its cognatereceptors, including EGF-R and HER4.

Certain antigen binding proteins described herein are antibodies or arederived from antibodies. In certain embodiments, the polypeptidestructure of the antigen binding proteins is based on antibodies,including, but not limited to, monoclonal antibodies, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to herein as “antibody mimetics”), chimericantibodies, humanized antibodies, human antibodies, antibody fusions(sometimes referred to herein as “antibody conjugates”), and fragmentsthereof, respectively. The various structures are further describedherein below.

The antigen binding proteins provided herein have been demonstrated tobind to several epitopes of HB-EGF, in particular human HB-EGF. Asdemonstrated in the examples, the ability of HB-EGF to bind to itscognate receptors is reduced or inhibited. As a consequence, the antigenbinding proteins provided herein are capable of inhibiting the activityof HB-EGF. In particular, antigen binding proteins binding to theseepitopes can have one or more of the following activities: inhibiting,inter alia, EGF-R and HER4 autophosphorylation, induction of EGF-R andHER4 signal transduction pathway, EGF-R and HER4 induced cell growth,and other physiological effects induced by EGF-R and HER4 upon HB-EGFbinding.

The antigen binding proteins that are disclosed herein have a variety ofutilities. Some of the antigen binding proteins, for instance, areuseful in specific binding assays, affinity purification of HB-EGF, inparticular hHB-EGF and in screening assays to identify pof suchreceptors. In addition, the disclosed antigen binding proteins may beused for the diagnosis and/or treatment of disease, such asproliferative disorders. These include, but are not limited to, varioustypes of cancer.

B. Heparin-Binding Epidermal Growth Factor-Like Growth Factor (HB-EGF)

HB-EGF is produced by various tumor cells and acts as an autocrine tumorgrowth factor. Davis-Fleischer et al., 1998, Front Biosci. 3:288-299;Iwamoto & Mekada, 2000, Cytokine Growth Factor Rev. 11:335-344. HB-EGFhas a strong affinity for heparin which can increase the biologicalactivity of HB-EGF. HB-EGF is produced as a transmembrane protein whichis proteolytically cleaved by metalloproteinases to yield the maturesoluble form of the growth factor.

HB-EGF was first identified from supernatants of cultured humanmacrophages in a soluble, secreted form. On human cells, the precursorproHB-EGF, acts as the diphtheria toxin receptor. Various cell types,including epithelial cells, keratinocytes, monocytes, mesangial cells,lymphoid cells, and skeletal muscle cells, produce HB-EGF. It is apotent mitogen and chemotactic factor for epithelial cells, fibroblasts,smooth muscle cells and various human cancer cells.

The transmembrane form of HB-EGF is synthesized by many cell types as a208-amino acid transmembrane precursor (tm-HB-EGF) containing EGF,heparin-binding, transmembrane, and cytoplasmic domains. Theextracellular domain can be released as a 12- to 22-kDa soluble form ofHB-EGF (sol-HB-EGF) through the action of metalloproteinases, which isregulated by different G protein-coupled receptors (GPCRs) or tumorpromoters such as tetradecanoyl phorbol acetate (TPA). Typically, asubstantial amount of transmembrane HB-EGF precursor remains uncleavedon the cell surface.

Both tm-HB-EGF and sol-HB-EGF are biologically active. The biologicalfunctions of both sol- and tm-HB-EGF are mediated by the EGF receptor(EGFR; HER1) and ErbB4 (HER4). Activation of these types of thesereceptors is believed to occur as a consequence of ligand-inducedreceptor homo- or hetero-dimerization. Upon activation, the EGF receptorhas been demonstrated to increase cell growth, increase cell motility,inhibit apoptosis and increase cellular transformation.

EGFR-dependent signaling pathways can be transactivated upon stimulationof G-protein-coupled receptors (GPCR). Ligand activation ofheterotrimeric G proteins by interaction with a GPCR results in anintracellular signal that induces the extracellular activity of atransmembrane metalloproteinase. Ligands that activate the GPCR pathwayinclude LPA (lysophosphatidic acid), thrombin, carbachol, bombesin, andendothelin. Such activation leads to extracellular processing of atransmembrane growth factor precursor and release of the mature factorwhich, directly or through the proteoglycan matrix, interacts with theectodomain of EGFR and activates it through tyrosine phosphorylation.See, Prenzel et al., 1999, Nature 402:884-888. Thus, HB-EGF is acomponent of a triple membrane-passing signal (TMPS) mechanism whereby aGPCR activates a membrane-bound metalloproteinase, which cleavesproHB-EGF to release the soluble growth factor, which subsequentlyactivates the EGF receptor. EGFR transactivation has been linked tovarious disease states such as cardiac hypertrophy (reviewed in Shah BH, Catt K J. Trends Pharmacol Sci. 2003 May; 24(5):239-244), vascularremodeling (reviewed in Eguchi et al., 2003, Biochem Soc Trans. 2003December; 31(Pt 6):1198-202.) and cancer (reviewed in Fischer et al.,2003, supra).

Sequences for HB-EGF proteins and nucleic acids encoding those proteinsare available to one of skill in the art. For example, such HB-EGFsequences can be found in the database provided by the National Centerfor Biotechnology Information (NCBI) (see,http://www.ncbi.nlm.nih.gov/). One example of a sequence for a HB-EGF isthe amino acid sequence at NCBI accession numbers NM 001945 andNP_(—)001936 (gi:4503413). This sequence is provided below for easyreference (SEQ ID NO:1072):

MKLLPSVVLKLFLAAVLSALVTGESLERLRRGLAAGTSNPDPPTVSTDQLLPLGGGRDRKVRDLQEADLDLLRVTLSSKPQALATPNKEEHGKRKKKGKGLGKKRDPCLRKYKDFCIHGECKYVKELRAPSCICHPGYHGERCHGLSLPVENRLYTYDHTTILAVVAVVLSSVCLLVIVGLLMFRYHRRG GYDVENEEKVKLGMTNSH.

Note that the HB-EGF sequence shown above (SEQ ID NO:1072) has thenineteen amino acid signal peptide (MKLLPSVVLK LFLAAVLSA, SEQ IDNO:1073).

The soluble extracellular domain consists of amino acids 1-149 of theabove HB-EGF sequence. This sequence for the HB-EGF solubleextracellular domain is provided below as SEQ ID NO:1074:

MKLLPSVVLKLFLAAVLSALVTGESLERLRRGLAAGTSNPDPPTVSTDQLLPLGGGRDRKVRDLQEADLDLLRVTLSSKPQALATPNKEEHGKRKKKGKGLGKKRDPCLRKYKDFCIHGECKYVKELRAPSCICHPGYHGERCHGLSLP.

Upon cleavage, a mature HB-EGF is generated that consists of amino acids63-149 (87 amino acids). This sequence for the mature HB-EGF is providedbelow as SEQ ID NO:1075:

DLQEADLDLLRVTLSSKPQALATPNKEEHGKRKKKGKGLGKKRDPCLRKYKDFCIHGECKYVKELRAPSCICHPGYHGERCHGLSLP.

HB-EGF interacts with and activates the epidermal growth factor receptor(EGFR). EGFR is a 170 kDa transmembrane glycoprotein consisting of anextracellular ligand-binding domain, a transmembrane region and anintracellular domain with tyrosine kinase activity. Binding of growthfactors to the EGFR results in internalization of the ligand-receptorcomplex, autophosphorylation of the receptor and other proteinsubstrates, leading ultimately to DNA synthesis and cell division. Theexternal ligand binding domain is not only stimulated by HB-EGF, butalso by EGF, TGFα and amphiregulin (AR).

Overexpression of the EGFR is often accompanied by the co-expression ofEGF-like growth factors, suggesting that an autocrine pathway forcontrol of growth may play a major part in the progression of tumors. Itis now widely believed that this is a mechanism by which tumor cells canescape normal physiological control.

C. HB-EGF Receptor Antigen Binding Proteins

A variety of selective binding agents useful for regulating the activityof HB-EGF are provided. These agents include, for instance, antigenbinding proteins that contain an antigen binding domain (e.g., singlechain antibodies, domain antibodies, immunoadhesions, and polypeptideswith an antigen binding region) and specifically bind to a HB-EGFpolypeptide, in particular human HB-EGF. Some of the agents, forexample, are useful in inhibiting the binding of HB-EGF to itsreceptors, and can thus be used to inhibit, interfere with, or modulateone or more activities associated with HB-EGF-mediated signaling.

In general, the antigen binding proteins that are provided typicallycomprise one or more CDRs as described herein (e.g., 1, 2, 3, 4, 5 or6). In some instances, the antigen binding protein comprises (a) apolypeptide structure and (b) one or more CDRs that are inserted intoand/or joined to the polypeptide structure. The polypeptide structurecan take a variety of different forms. For example, it can be, orcomprise, the framework of a naturally occurring antibody, or fragmentor variant thereof, or may be completely synthetic in nature. Examplesof various polypeptide structures are further described below.

In certain embodiments, the polypeptide structure of the antigen bindingproteins is an antibody or is derived from an antibody, including, butnot limited to, monoclonal antibodies, bispecific antibodies,minibodies, domain antibodies, synthetic antibodies (sometimes referredto herein as “antibody mimetics”), chimeric antibodies, humanizedantibodies, antibody fusions (sometimes referred to as “antibodyconjugates”), and portions or fragments of each, respectively. In someinstances, the antigen binding protein is an immunological fragment ofan antibody (e.g., a Fab, a Fab′, a F(ab′)₂, or a scFv). The variousstructures are further described and defined herein.

Certain of the antigen binding proteins as provided herein specificallybind to human HB-EGF. In a specific embodiment, the antigen bindingprotein specifically binds to human HB-EGF protein having the amino acidsequence of SEQ ID NO:1072.

In embodiments where the antigen binding protein is used for therapeuticapplications, an antigen binding protein can inhibit, interfere with ormodulate one or more biological activities of HB-EGF. In this case, anantigen binding protein binds specifically to and/or substantiallyinhibits binding of human HB-EGF to its receptor when an excess ofantibody reduces the quantity of human HB-EGF bound to its receptor, orvice versa, by at least about 20%, 40%, 60%, 80%, 85%, or more (forexample by measuring binding in an in vitro competitive binding assay).HB-EGF has many distinct biological effects, which can be measured inmany different assays in different cell types; examples of such assaysare provided herein.

1. Naturally Occurring Antibody Structure

Some of the antigen binding proteins that are provided have thestructure typically associated with naturally occurring antibodies. Thestructural units of these antibodies typically comprise one or moretetramers, each composed of two identical couplets of polypeptidechains, though some species of mammals also produce antibodies havingonly a single heavy chain. In a typical antibody, each pair or coupletincludes one full-length “light” chain (in certain embodiments, about 25kDa) and one full-length “heavy” chain (in certain embodiments, about50-70 kDa). Each individual immunoglobulin chain is composed of several“immunoglobulin domains”, each consisting of roughly 90 to 110 aminoacids and expressing a characteristic folding pattern. These domains arethe basic units of which antibody polypeptides are composed. Theamino-terminal portion of each chain typically includes a variabledomain that is responsible for antigen recognition. The carboxy-terminalportion is more conserved evolutionarily than the other end of the chainand is referred to as the “constant region” or “C region”. Human lightchains generally are classified as kappa and lambda light chains, andeach of these contains one variable domain and one constant domain.Heavy chains are typically classified as mu, delta, gamma, alpha, orepsilon chains, and these define the antibody's isotype as IgM, IgD,IgG, IgA, and IgE, respectively. IgG has several subtypes, including,but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes includeIgM1, and IgM2. IgA subtypes include IgA1 and IgA2. In humans, the IgAand IgD isotypes contain four heavy chains and four light chains; theIgG and IgE isotypes contain two heavy chains and two light chains; andthe IgM isotype contains five heavy chains and five light chains. Theheavy chain C region typically comprises one or more domains that may beresponsible for effector function. The number of heavy chain constantregion domains will depend on the isotype. IgG heavy chains, forexample, contain three C region domains known as C_(H)1, C_(H)2 andC_(H)3. The antibodies that are provided can have any of these isotypesand subtypes. In certain embodiments, the HB-EGF antibody is of theIgG1, IgG2, or IgG4 subtype.

In full-length light and heavy chains, the variable and constant regionsare joined by a “J” region of about twelve or more amino acids, with theheavy chain also including a “D” region of about ten more amino acids.See, e.g., Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989,New York: Raven Press (hereby incorporated by reference in its entiretyfor all purposes). The variable regions of each light/heavy chain pairtypically form the antigen binding site.

One example of a kappa Light Constant domain of an exemplary HB-EGFmonoclonal antibody has the amino acid sequence:

(SEQ ID NO: 187) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.

One example of an IgG2 heavy constant domain of an exemplary HB-EGFmonoclonal antibody has the amino acid sequence:

(SEQ ID NO: 188) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Variable regions of immunoglobulin chains generally exhibit the sameoverall structure, comprising relatively conserved framework regions(FR) joined by three hypervariable regions, more often called“complementarity determining regions” or CDRs. The CDRs from the twochains of each heavy chain/light chain pair mentioned above typicallyare aligned by the framework regions to form a structure that bindsspecifically to a specific epitope on the target protein (e.g., HB-EGF).From N-terminal to C-terminal, naturally-occurring light and heavy chainvariable regions both typically conform with the following order ofthese elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numberingsystem has been devised for assigning numbers to amino acids that occupypositions in each of these domains. This numbering system is defined inKabat Sequences of Proteins of Immunological Interest (1987 and 1991,NIH, Bethesda, Md.), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917;Chothia et al., 1989, Nature 342:878-883.

The various light chain and heavy chain variable regions provided hereinare depicted in FIGS. 1A-1P, and FIGS. 2A-2O, respectively. Each ofthese variable regions may be attached to the above heavy and lightchain constant regions to form a complete antibody heavy and lightchain, respectively. Further, each of the so generated heavy and lightchain sequences may be combined to form a complete antibody structure.

Specific examples of some of the full length light and heavy chains ofthe antibodies that are provided and their corresponding amino acidsequences are summarized in FIGS. 3A-3K and FIGS. 4A-4O, respectively.

Again, each of the exemplary light chains (U_(L)-1, U_(L)-2, U_(L)-3etc.) listed in FIGS. 3A-3K can be combined with any of the exemplaryheavy chains shown in FIGS. 4A-4O to form an antibody. Examples of suchcombinations include U_(L)-1 combined with any of U_(H)-1 throughU_(H)-58; U_(L)-2 combined with any of U_(H)-1 through U_(H)-58; orU_(L)-3 combined with any of U_(H)-1 through U_(H)-58, and so on. Insome instances, the antibodies include at least one light chain and oneheavy chain from those listed in FIGS. 3A-3K and FIGS. 4A-4O,respectively. In other instances, the antibodies contain two identicallight chains and two identical heavy chains. As an example, an antibodyor immunologically functional fragment may include two U_(L)-1 lightchains and two U_(H)-1 heavy chains, or two U_(L)-2 light chains and twoU_(H)-2 heavy chains, or two U_(L)-3 light chains and two U_(H)-3 heavychains and other similar combinations of pairs of light chains and pairsof heavy chains as listed in FIGS. 3A-3K and FIGS. 4A-4O, respectively.

Other antibodies that are provided are variants of antibodies formed bycombination of the heavy and light chains shown in FIGS. 3A-3K and FIGS.4A-4O, respectively, and comprise light and/or heavy chains that eachhave at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to theamino acid sequences of these chains. In some instances, such antibodiesinclude at least one light chain and one heavy chain, whereas in otherinstances the variant forms contain two identical light chains and twoidentical heavy chains.

2. Variable Domains of Antibodies

Also provided are antigen binding proteins that contain an antibodylight chain variable region selected from the group consisting ofU-V_(L)1, U-V_(L)2, U-V_(L)3, U-V_(L)4, U-V_(L)5, U-V_(L)6, U-V_(L)7,U-V_(L)8, U-V_(L)9, U-V_(L)10, U-V_(L)11, U-V_(L)12, U-V_(L)13,U-V_(L)14, U-V_(L)15, U-V_(L)16, U-V_(L)17, U-V_(L)18, U-V_(L)19,U-V_(L)20, U-V_(L)21, U-V_(L)22, U-V_(L)23, U-V_(L)24, U-V_(L)25,U-V_(L)26, U-V_(L)27, U-V_(L)28, U-V_(L)29, U-V_(L)30, U-V_(L)31,U-V_(L)32, U-V_(L)33, U-V_(L)34, U-V_(L)35, U-V_(L)36, U-V_(L)37,U-V_(L)38, U-V_(L)39, U-V_(L)40, U-V_(L)41, U-V_(L)42, U-V_(L)43,U-V_(L)44, U-V_(L)45, U-V_(L)46, U-V_(L)47, U-V_(L)48, U-V_(L)49,U-V_(L)50, U-V_(L)51, U-V_(L)52, U-V_(L)54, U-V_(L)55, U-V_(L)56,U-V_(L)57, U-V_(L)58, U-V_(L)59, U-V_(L)60, U-V_(L)61, U-V_(L)62,U-V_(L)64, and U-V_(L)65, and/or an antibody light chain variable regionselected from the group consisting of U-V_(H)1, U-V_(H)2, U-V_(H)3,U-V_(H)4, U-V_(H)5, U-V_(H)6, U-V_(H)7, U-V_(H)8, U-V_(H)9, U-V_(H)10,U-V_(H)11, U-V_(H)12, U-V_(H)13, U-V_(H)14, U-V_(H)15, U-V_(H)16,U-V_(H)17, U-V_(H)18, U-V_(H)19, U-V_(H)20, U-V_(H)21, U-V_(H)22,U-V_(H)23, U-V_(H)24, U-V_(H)25, U-V_(H)26, U-V_(H)27, U-V_(H)28,U-V_(H)29, U-V_(H)30, U-V_(H)31, U-V_(H)32, U-V_(H)33, U-V_(H)34,U-V_(H)35, U-V_(H)36, U-V_(H)37, U-V_(H)38, U-V_(H)39, U-V_(H)40,U-V_(H)41, U-V_(H)42, U-V_(H)43, U-V_(H)44, U-V_(H)45, U-V_(H)46,U-V_(H)47, U-V_(H)48, U-V_(H)49, U-V_(H)50, U-V_(H)51, U-V_(H)52,U-V_(H)53, U-V_(H)54, U-V_(H)55, U-V_(H)56, U-V_(H)57, and U-V_(H)58, asshown in FIGS. 1A-1P, and FIGS. 2A-2O, respectively, and immunologicallyfunctional fragments, derivatives, muteins and variants of these lightchain and heavy chain variable regions.

Sequence alignments of the various light and heavy chain variableregions, respectively, are provided in FIGS. 10A and 10B, and FIGS. 11Aand 11B, respectively.

Antigen binding proteins of this type can generally be designated by theformula “V_(H)x/V_(L)y,” where “x” corresponds to the number of heavychain variable regions and “y” corresponds to the number of the lightchain variable regions (in general, x and y are each 1 or 2).

Each of the light chain variable regions listed in FIGS. 1A-1P may becombined with any of the light chain variable regions shown in FIGS.2A-2O to form an antigen binding protein. Examples of such combinationsinclude U-V_(L)1 combined with any of U-V_(H)1, U-V_(H)2, U-V_(H)3,U-V_(H)4, U-V_(H)5, U-V_(H)6, U-V_(H)7, U-V_(H)8, U-V_(H)9, U-V_(H)10,U-V_(H)11, U-V_(H)12, U-V_(H)13, U-V_(H)14, U-V_(H)15, U-V_(H)16,U-V_(H)17, U-V_(H)18, U-V_(H)19, U-V_(H)20, U-V_(H)21, U-V_(H)22,U-V_(H)23, U-V_(H)24, U-V_(H)25, U-V_(H)26, U-V_(H)27, U-V_(H)28,U-V_(H)29, U-V_(H)30, U-V_(H)31, U-V_(H)32, U-V_(H)33, U-V_(H)34,U-V_(H)35, U-V_(H)36, U-V_(H)37, U-V_(H)38, U-V_(H)39, U-V_(H)40,U-V_(H)41, U-V_(H)42, U-V_(H)43, U-V_(H)44, U-V_(H)45, U-V_(H)46,U-V_(H)47, U-V_(H)48, U-V_(H)49, U-V_(H)50, U-V_(H)51, U-V_(H)52,U-V_(H)53, U-V_(H)54, U-V_(H)55, U-V_(H)56, U-V_(H)57, or U-V_(H)58, orU-V_(L)2 combined with any of U-V_(H)1, U-V_(H)2, U-V_(H)3, U-V_(H)4,U-V_(H)5, U-V_(H)6, U-V_(H)7, U-V_(H)8, U-V_(H)9, U-V_(H)10, U-V_(H)11,U-V_(H)12, U-V_(H)13, U-V_(H)14, U-V_(H)15, U-V_(H)16, U-V_(H)17,U-V_(H)18, U-V_(H)19, U-V_(H)20, U-V_(H)21, U-V_(H)22, U-V_(H)23,U-V_(H)24, U-V_(H)25, U-V_(H)26, U-V_(H)27, U-V_(H)28, U-V_(H)29,U-V_(H)30, U-V_(H)31, U-V_(H)32, U-V_(H)33, U-V_(H)34, U-V_(H)35,U-V_(H)36, U-V_(H)37, U-V_(H)38, U-V_(H)39, U-V_(H)40, U-V_(H)41,U-V_(H)42, U-V_(H)43, U-V_(H)44, U-V_(H)45, U-V_(H)46, U-V_(H)47,U-V_(H)48, U-V_(H)49, U-V_(H)50, U-V_(H)51, U-V_(H)52, U-V_(H)53,U-V_(H)54, U-V_(H)55, U-V_(H)56, U-V_(H)57, or U-V_(H)58, etc.

In some instances, the antigen binding protein includes at least oneheavy chain variable region and/or one light chain variable region fromthose listed in FIGS. 1A-1P, and FIGS. 2A-2O, respectively. In someinstances, the antigen binding protein includes at least two differentheavy chain variable regions and/or light chain variable regions fromthose listed in FIGS. 1A-1P, and FIGS. 2A-2O, respectively. An exampleof such an antigen binding protein comprises (a) one U-V_(L)1, and (b)one of U-V_(L)2, U-V_(L)3, U-V_(L)4, U-V_(L)5, U-V_(L)6, U-V_(L)7,U-V_(L)8, U-V_(L)9, U-V_(L)10, U-V_(L)11, U-V_(L)12, U-V_(L)13,U-V_(L)14, U-V_(L)15, U-V_(L)16, U-V_(L)17, U-V_(L)18, U-V_(L)19,U-V_(L)20, U-V_(L)21, U-V_(L)22, U-V_(L)23, U-V_(L)24, U-V_(L)25,U-V_(L)26, U-V_(L)27, U-V_(L)28, U-V_(L)29, U-V_(L)30, U-V_(L)31,U-V_(L)32, U-V_(L)33, U-V_(L)34, U-V_(L)35, U-V_(L)36, U-V_(L)37,U-V_(L)38, U-V_(L)39, U-V_(L)40, U-V_(L)41, U-V_(L)42, U-V_(L)43,U-V_(L)44, U-V_(L)45, U-V_(L)46, U-V_(L)47, U-V_(L)48, U-V_(L)49,U-V_(L)50, U-V_(L)51, U-V_(L)52, U-V_(L)54, U-V_(L)55, U-V_(L)56,U-V_(L)57, U-V_(L)58, U-V_(L)59, U-V_(L)60, U-V_(L)61, U-V_(L)62,U-V_(L)64, and U-V_(L)65. Again another example of such an antigenbinding protein comprises (a) one U-V_(L)2, and (b) one of U-V_(L)1,U-V_(L)3, U-V_(L)4, U-V_(L)5, U-V_(L)6, U-V_(L)7, U-V_(L)8, U-V_(L)9,U-V_(L)10, U-V_(L)11, U-V_(L)12, U-V_(L)13, U-V_(L)14, U-V_(L)15,U-V_(L)16, U-V_(L)17, U-V_(L)18, U-V_(L)19, U-V_(L)20, U-V_(L)21,U-V_(L)22, U-V_(L)23, U-V_(L)24, U-V_(L)25, U-V_(L)26, U-V_(L)27,U-V_(L)28, U-V_(L)29, U-V_(L)30, U-V_(L)31, U-V_(L)32, U-V_(L)33,U-V_(L)34, U-V_(L)35, U-V_(L)36, U-V_(L)37, U-V_(L)38, U-V_(L)39,U-V_(L)40, U-V_(L)41, U-V_(L)42, U-V_(L)43, U-V_(L)44, U-V_(L)45,U-V_(L)46, U-V_(L)47, U-V_(L)48, U-V_(L)49, U-V_(L)50, U-V_(L)51,U-V_(L)52, U-V_(L)54, U-V_(L)55, U-V_(L)56, U-V_(L)57, U-V_(L)58,U-V_(L)59, U-V_(L)60, U-V_(L)61, U-V_(L)62, U-V_(L)64, and U-V_(L)65.Again another example of such an antigen binding protein comprises (a)one U-V_(L)3, and (b) one of U-V_(L)1, U-V_(L)2, U-V_(L)4, U-V_(L)5,U-V_(L)6, U-V_(L)7, U-V_(L)8, U-V_(L)9, U-V_(L)10, U-V_(L)11, U-V_(L)12,U-V_(L)13, U-V_(L)14, U-V_(L)15, U-V_(L)16, U-V_(L)17, U-V_(L)18,U-V_(L)19, U-V_(L)20, U-V_(L)21, U-V_(L)22, U-V_(L)23, U-V_(L)24,U-V_(L)25, U-V_(L)26, U-V_(L)27, U-V_(L)28, U-V_(L)29, U-V_(L)30,U-V_(L)31, U-V_(L)32, U-V_(L)33, U-V_(L)34, U-V_(L)35, U-V_(L)36,U-V_(L)37, U-V_(L)38, U-V_(L)39, U-V_(L)40, U-V_(L)41, U-V_(L)42,U-V_(L)43, U-V_(L)44, U-V_(L)45, U-V_(L)46, U-V_(L)47, U-V_(L)48,U-V_(L)49, U-V_(L)50, U-V_(L)51, U-V_(L)52, U-V_(L)54, U-V_(L)55,U-V_(L)56, U-V_(L)57, U-V_(L)58, U-V_(L)59, U-V_(L)60, U-V_(L)61,U-V_(L)62, U-V_(L)64, and U-V_(L)65, etc.

Again another example of such an antigen binding protein comprises (a)one U-V_(H)1, and (b) one of U-V_(H)2, U-V_(H)3, U-V_(H)4, U-V_(H)5,U-V_(H)6, U-V_(H)7, U-V_(H)8, U-V_(H)9, U-V_(H)10, U-V_(H)11, U-V_(H)12,U-V_(H)13, U-V_(H)14, U-V_(H)15, U-V_(H)16, U-V_(H)17, U-V_(H)18,U-V_(H)19, U-V_(H)20, U-V_(H)21, U-V_(H)22, U-V_(H)23, U-V_(H)24,U-V_(H)25, U-V_(H)26, U-V_(H)27, U-V_(H)28, U-V_(H)29, U-V_(H)30,U-V_(H)31, U-V_(H)32, U-V_(H)33, U-V_(H)34, U-V_(H)35, U-V_(H)36,U-V_(H)37, U-V_(H)38, U-V_(H)39, U-V_(H)40, U-V_(H)41, U-V_(H)42,U-V_(H)43, U-V_(H)44, U-V_(H)45, U-V_(H)46, U-V_(H)47, U-V_(H)48,U-V_(H)49, U-V_(H)50, U-V_(H)51, U-V_(H)52, U-V_(H)53, U-V_(H)54,U-V_(H)55, U-V_(H)56, U-V_(H)57, and U-V_(H)58. Another examplecomprises (a) one U-V_(H)2, and (b) one of U-V_(H)1, U-V_(H)3, U-V_(H)4,U-V_(H)5, U-V_(H)6, U-V_(H)7, U-V_(H)8, U-V_(H)9, U-V_(H)10, U-V_(H)11,U-V_(H)12, U-V_(H)13, U-V_(H)14, U-V_(H)15, U-V_(H)16, U-V_(H)17,U-V_(H)18, U-V_(H)19, U-V_(H)20, U-V_(H)21, U-V_(H)22, U-V_(H)23,U-V_(H)24, U-V_(H)25, U-V_(H)26, U-V_(H)27, U-V_(H)28, U-V_(H)29,U-V_(H)30, U-V_(H)31, U-V_(H)32, U-V_(H)33, U-V_(H)34, U-V_(H)35,U-V_(H)36, U-V_(H)37, U-V_(H)38, U-V_(H)39, U-V_(H)40, U-V_(H)41,U-V_(H)42, U-V_(H)43, U-V_(H)44, U-V_(H)45, U-V_(H)46, U-V_(H)47,U-V_(H)48, U-V_(H)49, U-V_(H)50, U-V_(H)51, U-V_(H)52, U-V_(H)53,U-V_(H)54, U-V_(H)55, U-V_(H)56, U-V_(H)57, and U-V_(H)58. Again anotherexample comprises (a) one U-V_(H)3, and (b) one of U-V_(H)1, U-V_(H)2,U-V_(H)4, U-V_(H)5, U-V_(H)6, U-V_(H)7, U-V_(H)8, U-V_(H)9, U-V_(H)10,U-V_(H)11, U-V_(H)12, U-V_(H)13, U-V_(H)14, U-V_(H)15, U-V_(H)16,U-V_(H)17, U-V_(H)18, U-V_(H)19, U-V_(H)20, U-V_(H)21, U-V_(H)22,U-V_(H)23, U-V_(H)24, U-V_(H)25, U-V_(H)26, U-V_(H)27, U-V_(H)28,U-V_(H)29, U-V_(H)30, U-V_(H)31, U-V_(H)32, U-V_(H)33, U-V_(H)34,U-V_(H)35, U-V_(H)36, U-V_(H)37, U-V_(H)38, U-V_(H)39, U-V_(H)40,U-V_(H)41, U-V_(H)42, U-V_(H)43, U-V_(H)44, U-V_(H)45, U-V_(H)46,U-V_(H)47, U-V_(H)48, U-V_(H)49, U-V_(H)50, U-V_(H)51, U-V_(H)52,U-V_(H)53, U-V_(H)54, U-V_(H)55, U-V_(H)56, U-V_(H)57, and U-V_(H)58,etc.

The various combinations of heavy chain variable regions may be combinedwith any of the various combinations of light chain variable regions.

In other instances, the antigen binding protein contains two identicallight chain variable regions and/or two identical heavy chain variableregions. As an example, the antigen binding protein may be an antibodyor immunologically functional fragment that includes two light chainvariable regions and two heavy chain variable regions in combinations ofpairs of light chain variable regions and pairs of heavy chain variableregions as listed in FIGS. 1A-1P, and FIGS. 2A-2O, respectively.

Some antigen binding proteins that are provided comprise a light chainvariable domain comprising a sequence of amino acids that differs fromthe sequence of a light chain variable domain selected from U-V_(L)1,U-V_(L)2, U-V_(L)3, U-V_(L)4, U-V_(L)5, U-V_(L)6, U-V_(L)7, U-V_(L)8,U-V_(L)9, U-V_(L)10, U-V_(L)11, U-V_(L)12, U-V_(L)13, U-V_(L)14,U-V_(L)15, U-V_(L)16, U-V_(L)17, U-V_(L)18, U-V_(L)19, U-V_(L)20,U-V_(L)21, U-V_(L)22, U-V_(L)23, U-V_(L)24, U-V_(L)25, U-V_(L)26,U-V_(L)27, U-V_(L)28, U-V_(L)29, U-V_(L)30, U-V_(L)31, U-V_(L)32,U-V_(L)33, U-V_(L)34, U-V_(L)35, U-V_(L)36, U-V_(L)37, U-V_(L)38,U-V_(L)39, U-V_(L)40, U-V_(L)41, U-V_(L)42, U-V_(L)43, U-V_(L)44,U-V_(L)45, U-V_(L)46, U-V_(L)47, U-V_(L)48, U-V_(L)49, U-V_(L)50,U-V_(L)51, U-V_(L)52, U-V_(L)54, U-V_(L)55, U-V_(L)56, U-V_(L)57,U-V_(L)58, U-V_(L)59, U-V_(L)60, U-V_(L)61, U-V_(L)62, U-V_(L)64, orU-V_(L)65 at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15amino acid residues, wherein each such sequence difference isindependently either a deletion, insertion or substitution of one aminoacid. The light chain variable region in some antigen binding proteinscomprises a sequence of amino acids that has at least 70%, 75%, 80%,85%, 90%, 95%, 97% or 99% sequence identity to the amino acid sequencesof the light chain variable region of U-V_(L)1, U-V_(L)2, U-V_(L)3,U-V_(L)4, U-V_(A)5, U-V_(L)6, U-V_(L)7, U-V_(L)8, U-V_(L)9, U-V_(L)10,U-V_(L)11, U-V_(L)12, U-V_(L)13, U-V_(L)14, U-V_(L)15, U-V_(L)16,U-V_(L)17, U-V_(L)18, U-V_(L)19, U-V_(L)20, U-V_(L)21, U-V_(L)22,U-V_(L)23, U-V_(L)24, U-V_(L)25, U-V_(L)26, U-V_(L)27, U-V_(L)28,U-V_(L)29, U-V_(L)30, U-V_(L)31, U-V_(L)32, U-V_(L)33, U-V_(L)34,U-V_(L)35, U-V_(L)36, U-V_(L)37, U-V_(L)38, U-V_(L)39, U-V_(L)40,U-V_(L)41, U-V_(L)42, U-V_(L)43, U-V_(L)44, U-V_(L)45, U-V_(L)46,U-V_(L)47, U-V_(L)48, U-V_(L)49, U-V_(L)50, U-V_(L)51, U-V_(L)52,U-V_(L)54, U-V_(L)55, U-V_(L)56, U-V_(L)57, U-V_(L)58, U-V_(L)59,U-V_(L)60, U-V_(L)61, U-V_(L)62, U-V_(L)64, or U-V_(L)65.

Certain antibodies comprise a heavy chain variable domain comprising asequence of amino acids that differs from the sequence of a heavy chainvariable domain selected from U-V_(H)1, U-V_(H)2, U-V_(H)3, U-V_(H)4,U-V_(H)5, U-V_(H)6, U-V_(H)7, U-V_(H)8, U-V_(H)9, U-V_(H)10, U-V_(H)11,U-V_(H)12, U-V_(H)13, U-V_(H)14, U-V_(H)15, U-V_(H)16, U-V_(H)17,U-V_(H)18, U-V_(H)19, U-V_(H)20, U-V_(H)21, U-V_(H)22, U-V_(H)23,U-V_(H)24, U-V_(H)25, U-V_(H)26, U-V_(H)27, U-V_(H)28, U-V_(H)29,U-V_(H)30, U-V_(H)31, U-V_(H)32, U-V_(H)33, U-V_(H)34, U-V_(H)35,U-V_(H)36, U-V_(H)37, U-V_(H)38, U-V_(H)39, U-V_(H)40, U-V_(H)41,U-V_(H)42, U-V_(H)43, U-V_(H)44, U-V_(H)45, U-V_(H)46, U-V_(H)47,U-V_(H)48, U-V_(H)49, U-V_(H)50, U-V_(H)51, U-V_(H)52, U-V_(H)53,U-V_(H)54, U-V_(H)55, U-V_(H)56, U-V_(H)57, or U-V_(H)58 at only 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues,wherein each such sequence difference is independently either adeletion, insertion or substitution of one amino acid. The heavy chainvariable region in some antigen binding proteins comprises a sequence ofamino acids that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99%sequence identity to the amino acid sequences of the heavy chainvariable region of U-V_(H)1, U-V_(H)2, U-V_(H)3, U-V_(H)4, U-V_(H)5,U-V_(H)6, U-V_(H)7, U-V_(H)8, U-V_(H)9, U-V_(H)10, U-V_(H)11, U-V_(H)12,U-V_(H)13, U-V_(H)14, U-V_(H)15, U-V_(H)16, U-V_(H)17, U-V_(H)18,U-V_(H)19, U-V_(H)20, U-V_(H)21, U-V_(H)22, U-V_(H)23, U-V_(H)24,U-V_(H)25, U-V_(H)26, U-V_(H)27, U-V_(H)28, U-V_(H)29, U-V_(H)30,U-V_(H)31, U-V_(H)32, U-V_(H)33, U-V_(H)34, U-V_(H)35, U-V_(H)36,U-V_(H)37, U-V_(H)38, U-V_(H)39, U-V_(H)40, U-V_(H)41, U-V_(H)42,U-V_(H)43, U-V_(H)44, U-V_(H)45, U-V_(H)46, U-V_(H)47, U-V_(H)48,U-V_(H)49, U-V_(H)50, U-V_(H)51, U-V_(H)52, U-V_(H)53, U-V_(H)54,U-V_(H)55, U-V_(H)56, U-V_(H)57, or U-V_(H)58.

Still other antigen binding proteins, e.g., antibodies orimmunologically functional fragments include variant forms of a variantlight chain and a variant heavy chain as just described.

3. CDRs

The antigen binding proteins disclosed herein are polypeptides intowhich one or more CDRs are grafted, inserted and/or joined. An antigenbinding protein can have 1, 2, 3, 4, 5 or 6 CDRs. An antigen bindingprotein thus can have, for example, one light chain CDR1 (“CDRL1”),and/or one light chain CDR2 (“CDRL2”), and/or one light chain CDR3(“CDRL3”), and/or one heavy chain CDR1 (“CDRH1”), and/or one heavy chainCDR2 (“CDRH2”), and/or one heavy chain CDR3 (“CDRH3”). Some antigenbinding proteins include both a CDRL3 and a CDRH3. Specific CDRs areidentified in FIGS. 6A-6F, and FIGS. 7A-7E.

Complementarity determining regions (CDRs) and framework regions (FR)(examples of light and heavy chain FR amino acid sequences are given inFIGS. 8A-8H and FIGS. 9A-9F, respectively) of a given antibody may beidentified using the system described by Kabat et al. in Sequences ofProteins of Immunological Interest, 5th Ed., US Dept. of Health andHuman Services, PHS, NIH, NIH Publication No. 91-3242, 1991. Certainantibodies that are disclosed herein comprise one or more amino acidsequences that are identical or have substantial sequence identity tothe amino acid sequences of one or more of the CDRs presented in FIGS.6A-6F (CDRLs) and FIGS. 7A-7E (CDRHs).

The structure and properties of CDRs within a naturally occurringantibody has been described, supra. Briefly, in a traditional antibody,the CDRs are embedded within a framework in the heavy and light chainvariable region where they constitute the regions responsible forantigen binding and recognition. A variable region comprises at leastthree heavy or light chain CDRs, see, supra (Kabat et al., 1991,Sequences of Proteins of Immunological Interest, Public Health ServiceN.I.H., Bethesda, Md.; see, also Chothia and Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342: 877-883), within aframework region (designated framework regions 1-4, FR1, FR2, FR3, andFR4, by Kabat et al., 1991, supra; see, also Chothia and Lesk, 1987,supra). The CDRs provided herein, however, may not only be used todefine the antigen binding domain of a traditional antibody structure,but may be embedded in a variety of other polypeptide structures, asdescribed herein.

In one aspect, the CDRs provided are a (a) a CDRL selected from thegroup consisting of (i) a CDRL1 selected from the group consisting ofSEQ ID NOs:189-217; (ii) a CDRL2 selected from the group consisting ofSEQ ID NO:218-233; (iii) a CDRL3 selected from the group consisting ofSEQ ID NO:234-274; and (iv) a CDRL of (i), (ii) and (iii) that containsone or more amino acid substitutions, deletions or insertions of no morethan five, four, three, two, or one amino acids; (B) a CDRH selectedfrom the group consisting of (i) a CDRH1 selected from the groupconsisting of SEQ ID NO:275-299; (ii) a CDRH2 selected from the groupconsisting of SEQ ID NO:300-331; (iii) a CDRH3 selected from the groupconsisting of SEQ ID NO:332-372; and (iv) a CDRLH of (i), (ii) and (iii)that contains one or more amino acid substitutions, deletions orinsertions of no more than five, four, three, two, or one amino acidsamino acids.

In yet another aspect, variant forms of the CDRs are provided that haveat least 80%, 85%, 90% or 95% sequence identity to a CDR sequence listedin FIGS. 6A-6F and FIGS. 7A-7E.

In yet another aspect, the CDRs disclosed herein include consensussequences derived from groups of related monoclonal antibodies. Asdescribed herein, a “consensus sequence” refers to amino acid sequenceshaving conserved amino acids common among a number of sequences andvariable amino acids that vary within a given amino acid sequences. TheCDR consensus sequences provided include CDRs corresponding to each ofCDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3.

Consensus sequences were determined using a standard phylogenic analysisapproach of the CDRs corresponding to the U-V_(L) and U-V_(H) ofanti-HB-EGF antibodies. First, in this approach, amino acid sequencescorresponding to the entire variable domains of either U-V_(L) orU-V_(H) were converted to FASTA formatting for ease in processingcomparative alignments and inferring phylogenies. Based on thiscomparison, each the light and heavy chain variable regions,respectively were divided in phylogenetically related groups, i.e., thelight chain variable regions were divided into six groups A, B, C, D, E,and F (see, FIGS. 12A and 12B), and the heavy chain variable regionswere divided into seven groups A, B, C, D, E, F, and G (see, FIG. 12C).Then, within each of these groups, comparison of each of the CDRL1,CDRL2, CDRL3, CDRH1, CDRH2, CDRH3 regions was used to define consensuscollections.

Group A of the light chain CDRs includes the following consensuscollections:

a. a CDRL1 of the generic formula X₁SSQSLX₂X₃SDGX₄TYLX₅ (SEQ IDNO:1035), wherein

-   -   X₁ is K or R,    -   X₂ is L or V,    -   X₃ is H or Y,    -   X₄ is K or N,    -   X₅ is N, S or Y.

b. a CDRL2 of the generic formula X₁X₂SNX₃X₄S (SEQ ID NO:1041), wherein

-   -   X₁ is E or K,    -   X₂ is I or V,    -   X₃ is R or W,    -   X₄ is D or F.

c. a CDRL3 of the generic formula X₁QX₂X₃X₄X₅PX₆X₇ (SEQ ID NO:1046),wherein

-   -   X₁ is I or M,    -   X₂ is A, G or S,    -   X₃ is I or T,    -   X₄ is H or Q,    -   X₅ is F, L or W,    -   X₆ is C, I, H, L or T,    -   X₇ is S or T.

Group B of the light chain CDRs includes the following consensuscollections:

a. a CDRL1 of the generic formula RASQX₁₁SX₂YLN (SEQ ID NO:1036),wherein

-   -   X₁ is R, S or T,    -   X₂ is R or S.

b. a CDRL2 of the generic formula X₁X₂SX₃LQS (SEQ ID NO:1042), wherein

-   -   X₁ is A or T,    -   X₂ is A, E or V,    -   X₃ is S or T.

c. a CDRL3 of the generic formula QQX₁X₂X₃X₄X₅IT (SEQ ID NO:1047),wherein

-   -   X₁ is I or S,    -   X₂ is F or Y,    -   X₃ is F, I, S or Y,    -   X₄ is A, S or T,    -   X₅ is P or S.

Group C of the light chain CDRs includes the following consensuscollections:

a. a CDRL1 of the generic formula RASQX₁IX₂X₃X₄LX₅ (SEQ ID NO:1037),wherein

-   -   X₁ is D, G, S or T,    -   X₂ is A, R or S,    -   X₃ is H, I, N, R, S or T,    -   X₄ is D, W or Y,    -   X₅ is A, G or N.

b. a CDRL2 of the generic formula X₁ASX₂LQS (SEQ ID NO:1043), wherein

-   -   X₁ is A or V,    -   X₂ is S or T.

c. a CDRL3 of the generic formula X₁X₂X₃X₄X₅X₆X₇X₈T (SEQ ID NO:1048),wherein

-   -   X₁ is L or Q,    -   X₂ is K, N or Q,    -   X₃ is A, H, S or Y,    -   X₄ is H, N or Y,    -   X₅ is N, S or T,    -   X₆ is A, F, I, T, V or Y,    -   X₇ is P or no amino acid,    -   X₈ is F, L or P.

Group D of the light chain CDRs includes the following consensuscollections:

a. a CDRL1 of the generic formula QASQDIX₁X₂X₃LN (SEQ ID NO:1038),wherein

-   -   X₁ is S or T,    -   X₂ is D or N,    -   X₃ is S or Y.

b. a CDRL2 of the generic formula DASX₁LET (SEQ ID NO:1044), wherein

-   -   X₁ is I or N.

c. a CDRL3 of the generic formula QX₁X₂DX₃LPX₄X₅ (SEQ ID NO:1049),wherein

-   -   X₁ is H or Q,    -   X₂ is C or Y,    -   X₃ is D, I, N, S or Y,    -   X₄ is F, I or L,    -   X₅ is A, S or T.

Group E of the light chain CDRs includes the following consensuscollections:

a. a CDRL1 of the generic formula RASQX₁VX₂X₃X₄X₅LA (SEQ ID NO:1039),wherein

-   -   X₁ is S or T,    -   X₂ is I or S,    -   X₃ is R or S,    -   X₄ is S, N or no amino acid,    -   X₅ is Y or no amino acid.

b. a CDRL2 of the generic formula GASSRAT (SEQ ID NO:223)

c. a CDRL3 of the generic formula QQX₁X₂X₃X₄PX₅X₆X₇ (SEQ ID NO:1050),wherein

-   -   X₁ is H or Y,    -   X₂ is G or N,    -   X₃ is N or S,    -   X₄ is S or W,    -   X₅ is P or no amino acid,    -   X₆ is R or W,    -   X₇ is S or T.

Group F of the light chain CDRs includes the following consensuscollections:

a. a CDRL1 of the generic formula KSSQX₁X₂LX₃X₄SNNKNYLX₅ (SEQ IDNO:1040), wherein

-   -   X₁ is N or S,    -   X₂ is I or V,    -   X₃ is D or Y,    -   X₄ is N, R or S,    -   X₅ is A or V.

b. a CDRL2 of the generic formula WASX₁RES (SEQ ID NO:1045), wherein

-   -   X₁ is A or T.

c. a CDRL3 of the generic formula X₁QYX₂X₃X₄X₅X₆X₇F (SEQ ID NO:1051),wherein

-   -   X₁ is H or Q,    -   X₂ is F or Y,    -   X₃ is G, I or S,    -   X₄ is F, I or T,    -   X₅ is M, P, S or T,    -   X₆ is F, L, R or W,    -   X₇ is S or T

Group A of the heavy chain CDRs includes the following consensuscollections:

a. a CDRH1 of the generic formula GYTX₁TX₂X₃X₄X₅X₆ (SEQ ID NO:1052),wherein

-   -   X₁ is F or L,    -   X₂ is E, G or S,    -   X₃ is H, L or Y,    -   X₄ is G, S or Y,    -   X₅ is I or M,    -   X₆ is H or S.

b. a CDRH2 of the generic formula X₁X₂X₃X₄X₅X₆GX₇TX₈X₉X₁₀QKX₁₁X₁₂ (SEQID NO:1058), wherein

-   -   X₁ is S or W,    -   X₂ is F or I,    -   X₃ is D, N or S,    -   X₄ is A, P,    -   X₅ is E, N or S,    -   X₆ is D, N or S,    -   X₇ is E, G or N,    -   X₈ is I or N,    -   X₉ is C, H or Y,    -   X₁₀ is A or T,    -   X₁₁ is F or L,    -   X₁₂ is D or G.

c. a CDRH3 of the generic formula X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁DX₁₂ (SEQ IDNO:1065), wherein

-   -   X₁ is E or S,    -   X₂ is D, G or no amino acid,    -   X₃ is D, N or no amino acid,    -   X₄ is G or no amino acid,    -   X₅ is G or no amino acid,    -   X₆ is W, Y or no amino acid,    -   X₇ is I, N or Y,    -   X₈ is A or Y,    -   X₉ is G, V or Y,    -   X₁₀ is A, F or G,    -   X₁₁ is F, L or M,    -   X₁₂ is V or Y.

Group B of the heavy chain CDRs includes the following consensuscollections:

a. a CDRH1 of the generic formula GYX₁FTSYWIG (SEQ ID NO:1053), wherein

-   -   X₁ is R or S.

b. a CDRH2 of the generic formula IIYPX₁DSDX₂RYSPSFQG (SEQ ID NO:1059),wherein

-   -   X₁ is D or G,    -   X₂ is A, I or T.

c. a CDRH3 of the generic formulaQX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁YX₁₂X₁₃X₁₄DX₁₅ (SEQ ID NO:1066), wherein

-   -   X₁ is G or no amino acid,    -   X₂ is K, L or Y,    -   X₃ is A, G or S,    -   X₄ is S, V or Y,    -   X₅ is A or G,    -   X₆ is G or no amino acid,    -   X₇ is T or no amino acid,    -   X₈ is S or no amino acid,    -   X₉ is Y or no amino acid,    -   X₁₀ is W or Y,    -   X₁₁ is G, S or Y,    -   X₁₂ is F or Y,    -   X₁₃ is G or no amino acid,    -   X₁₄ is M or no amino acid,    -   X₁₅ is V or Y.

Group C of the heavy chain CDRs includes the following consensuscollections:

a. a CDRH1 of the generic formula GFTFX₁SX₂X₃MH (SEQ ID NO:1054),wherein

-   -   X₁ is R or S,    -   X₂ is H or Y,    -   X₃ is D or G.

b. a CDRH2 of the generic formula X₁IX₂X₃DGSX₄X₅X₆YX₇DSVX₈G (SEQ IDNO:1060), wherein

-   -   X₁ is F or V,    -   X₂ is S or W,    -   X₃ is D, S or Y,    -   X₄ is I, N or T,    -   X₅ is K or Q,    -   X₆ is N, R or Y,    -   X₇ is A, T or V,    -   X₈ is K or R.

c. a CDRH3 of the generic formula X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQID NO:1067), wherein

-   -   X₁ is D, G, L, S or no amino acid,    -   X₂ is G, H, W, Y or no amino acid,    -   X₃ is A, F, W, Y or no amino acid,    -   X₄ is D, G, Q, T or no amino acid,    -   X₅ is G, I, Q, S or no amino acid,    -   X₆ is A, D, N, Q, S or no amino acid,    -   X₇ is G, Y or no amino acid,    -   X₈ is D, Y or no amino acid,    -   X₉ is Y or no amino acid,    -   X₁₀ is A, E, N or Y,    -   X₁₁ is G, P, T, V or Y,    -   X₁₂ is F or I,    -   X₁₃ is D or Q,    -   X₁₄ is C, H, V or Y.

Group D of the heavy chain CDRs includes the following consensuscollections:

a. a CDRH1 of the generic formula GFX₁FSX₂YX₃MX₄ (SEQ ID NO:1055),wherein

-   -   X₁ is P or T,    -   X₂ is A, R or S,    -   X₃ is A or S,    -   X₄ is N or S.

b. a CDRH2 of the generic formula X₁₁SX₂SX₃X₄X₅X₆YYADSVKG (SEQ IDNO:1061), wherein

-   -   X₁ is A, H or Y,    -   X₂ is G, R or S,    -   X₃ is G or S,    -   X₄ is G, R or S,    -   X₅ is S, T or Y,    -   X₆ is I or T.

c. a CDRH3 of the generic formulaX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇DX₁₈ (SEQ ID NO:1068), wherein

-   -   X₁ is E, D or no amino acid,    -   X₂ is G, R or no amino acid,    -   X₃ is I, V, Y or no amino acid,    -   X₄ is A, G, L or N,    -   X₅ is A, G, V or W,    -   X₆ is A, N, R or T,    -   X₇ is G, N, P or no amino acid,    -   X₈ is G, T, no amino acid,    -   X₉ is A or no amino acid,    -   X₁₀ is D, E or no amino acid,    -   X₁₁ is S, Y or no amino acid,    -   X₁₂ is G, Y or no amino acid,    -   X₁₃ is N, Y or no amino acid,    -   X₁₄ is Y or no amino acid,    -   X₁₅ is D, Y or no amino acid,    -   X₁₆ is A, G or no amino acid,    -   X₁₇ is F or M,    -   X₁₈ is I, V or Y.

Group E of the heavy chain CDRs includes the following consensuscollections:

-   -   a. a CDRH1 of the generic formula GX₁SX₂SX₃X₄X₅X₆X₇WX₈ (SEQ ID        NO:1056), wherein    -   X₁ is D or G,    -   X₂ is F, I or V,    -   X₃ is R, S or no amino acid,    -   X₄ is G, Y or no amino acid,    -   X₅ is D, G, S or no amino acid,    -   X₆ is A, S or Y,    -   X₇ is A or Y,    -   X₈ is N or S.

b. a CDRH2 of the generic formula X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁YX₁₂X₁₃SX₁₄KS(SEQ ID NO:1062), wherein

-   -   X₁ is E, R or Y,    -   X₂ is I or T,    -   X₃ is H, N or Y,    -   X₄ is C, H, S, T or Y,    -   X₅ is S or R,    -   X₆ is G or S,    -   X₇ is G, K, S or T,    -   X₈ is T or W,    -   X₉ is N or Y,    -   X₁₀ is N or no amino acid,    -   X₁₁ is D or no amino acid,    -   X₁₂ is A or N,    -   X₁₃ is P or V,    -   X₁₄ is L or V.

c. a CDRH3 of the generic formula

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃ (SEQ IDNO:1069), wherein

-   -   X₁ is A, D, G, S or T,    -   X₂ is A, E, G, L, N, R, Y or no amino acid,    -   X₃ is A, G, L, N, R, T, Y or no amino acid,    -   X₄ is D, G, R, S, V, Y or no amino acid,    -   X₅ is A, G, I, S, V, Y or no amino acid,    -   X₆ is F, G, L, R, V or no amino acid,    -   X₇ is L, T, Y or no amino acid,    -   X₈ is Y or no amino acid,    -   X₉ is Y or no amino acid,    -   X₁₀ is D or no amino acid,    -   X₁₁ is S or no amino acid,    -   X₁₂ is S or no amino acid,    -   X₁₃ is G or no amino acid,    -   X₁₄ is D, L, M, S, Y or no amino acid,    -   X₁₅ is H, I, P, V, W or no amino acid,    -   X₁₆ is F, G, L, R, S, Y or no amino acid,    -   X₁₇ is D, F, V, W, Y or no amino acid,    -   X₁₈ is C, F, L, P, S or Y,    -   X₁₉ is D, F, G or Y,    -   X₂₀ is A, C, G, P, R, V or Y,    -   X₂₁ is F, L, M, S or no amino acid,    -   X₂₂ is A, D or no amino acid,    -   X₂₃ is I, L, V, Y or no amino acid.

Group F of the heavy chain CDRs includes the following consensuscollections:

a. a CDRH1 of the generic formula GFSLSNARMGVS (SEQ ID NO:279).

b. a CDRH2 of the generic formula X₁IFSNDEKSYSTSLKS (SEQ ID NO:1063),wherein

-   -   X₁ is H or L.

c. a CDRH3 of the generic formula X₁YSSGWX₂X₃YGX₄X₅DX₆ (SEQ ID NO:1070),wherein

-   -   X₁ is M or V,    -   X₂ is S or no amino acid,    -   X₃ is F or no amino acid,    -   X₄ is V or no amino acid,    -   X₅ is F or M,    -   X₆ is V or Y.

Group G of the heavy chain CDRs includes the following consensuscollections:

a. a CDRH1 of the generic formula GFSLX₁TGGVGVG (SEQ ID NO:1057),wherein

-   -   X₁ is S or N.

b. a CDRH2 of the generic formula LIYWNX₁X₂KRYSPSLX₃S (SEQ ID NO:1064),wherein

-   -   X₁ is D or V,    -   X₂ is D or E,    -   X₃ is K or R.

c. a CDRH3 of the generic formula RX₁X₂X₃PFX₄Y (SEQ ID NO:1071), wherein

-   -   X₁ is G, H, L, N or R,    -   X₂ is E, T or W,    -   X₃ is L, N, T or V,    -   X₄ is D or E.

In another approach, consensus sequences may be determined by keepingthe CDRs contiguous within the same sequence corresponding to a U-V_(L)or U-V_(H). Briefly, in this approach, amino acid sequencescorresponding to the entire variable domains of either U-V_(L) orU-V_(H) are converted to FASTA formatting for ease in processingcomparative alignments and inferring phylogenies. Next, frameworkregions of these sequences are replaced with an artificial linkersequence so that examination of the CDRs alone is performed withoutintroducing any amino acid position weighting bias due to coincidentevents (e.g., such as unrelated antibodies that serendipitously share acommon germline framework heritage) whilst still keeping CDRs contiguouswithin the same sequence corresponding to a U-V, or U-V_(H). U-V_(L) orU-V_(H) sequences of this format are then subjected to sequencesimilarity alignment interrogation using a program that employs astandard ClutalW-like algorithm (see, Thompson et al., 1994, NucleicAcids Res. 22:4673-4680). This program likewise generates phylograms(phylogenic tree illustrations) based on sequence similarity alignmentsusing either UPGMA (unweighted pair group method using arithmeticaverages) or Neighbor-Joining methods (see, Saitou and Nei, 1987,Molecular Biology and Evolution 4:406-425) to construct and illustratesimilarity and distinction of sequence groups via branch lengthcomparison and grouping. Both methods produce similar results todetermine consensus sequence collections within the individual groups.

In some cases the antigen binding protein comprises at least one CDRL1,CDRL2, or CDRL3 having one of the above consensus sequences. In somecases, the antigen binding protein comprises at least one CDRH1, CDRH2,or CDRH3 having one of the above consensus sequences. In other cases,the antigen binding protein comprises at least two CDRLs according tothe above consensus sequences, and/or at least two CDRHs according tothe above consensus sequences. In one aspect, the CDRLs and/or CDRHs arederived from different groups. In other cases, the antigen bindingprotein comprises at least two CDRLs from the same group A, B, C, D, E,or F and/or at least two CDRHs from the same group A, B, C, D, E, F, orG. In other aspects, the antigen binding protein comprises all threeCDRL1, CDRL2, and CDRL3 sequences from the same of the above groups A,B, C, D, E, or F, and/or all three CDRH1, CDRH2, and CDRH3 sequence fromthe same of the above groups A, B, C, D, E, F, or G.

D. Exemplary Antigen Binding Proteins

According to one aspect, an isolated antigen binding protein is providedthat binds HB-EGF comprising (A) one or more light chain complementarydetermining regions (CDRLs) selected from the group consisting of: (i) aCDRL1 selected from the group consisting of SEQ ID NO:189-217; (ii) aCDRL2 selected from the group consisting of SEQ ID NO:218-233; (iii) aCDRL3 selected from the group consisting of SEQ ID NO:234-274; and (iv)a CDRL of (i), (ii) and (iii) that contains one or more amino acidsubstitutions, deletions or insertions of no more than five, four,three, four, two or one amino acids; (B) one or more heavy chaincomplementary determining regions (CDRHs) selected from the groupconsisting of: (i) a CDRH1 selected from the group consisting of SEQ IDNO:275-299; (ii) a CDRH2 selected from the group consisting of SEQ IDNO:300-331; (iii) a CDRH3 selected from the group consisting of SEQ IDNO:332-372; and (iv) a CDRH of (i), (ii) and (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more thanfive, four, three, four, two or one amino acids; or (C) one or morelight chain CDRLs of (A); and (D) one or more heavy chain CDRHs of (B).

In yet another embodiment, the isolated antigen binding protein maycomprise (A) a CDRL selected from the group consisting of (i) a CDRL1selected from the group consisting of SEQ ID NO:189-217; (ii) a CDRL2selected from the group consisting of SEQ ID NO:218-233; and (iii) aCDRL3 selected from the group consisting of SEQ ID NO:234-274; (B) aCDRH selected from the group consisting of (i) a CDRH1 selected from thegroup consisting of SEQ ID NO:275-299; (ii) a CDRH2 selected from thegroup consisting of SEQ ID NO:300-331; and (iii) a CDRH3 selected fromthe group consisting of SEQ ID NO:332-372; or (C) one or more lightchain CDRLs of (A); and (D) one or more heavy chain CDRLs of (B). In oneembodiment, the isolated antigen binding protein may include (A) a CDRL1of SEQ ID NO:189-217, a CDRL2 of SEQ ID NO:218-233, and a CDRL3 of SEQID NO:234-274, and (B) a CDRH1 of SEQ ID NO:275-299, a CDRH2 of SEQ IDNO:300-331, and a CDRH3 of SEQ ID NO:332-372.

In another embodiment, the antigen binding protein comprises a variablelight chain (V_(L)) has at least 80%, 85%, 90% or 95% sequence identitywith an amino acid sequence selected from the group consisting of SEQ IDNO:94-141, and/or the variable heavy chain (V_(H)) has at least 80%,85%, 90% or 95% sequence identity with an amino acid sequence selectedfrom the group consisting of SEQ ID NO:142-186. In a further embodiment,the V_(L) is selected from the group consisting of SEQ ID NO:94-141,and/or the V_(H) is selected from the group consisting of SEQ IDNO:142-186.

In another aspect, also provided is an isolated antigen binding proteinthat specifically binds to an epitope containing at least oneIHGE-containing epitope and/or EGF-like epitope of HB-EGF.

In a further aspect, there is a provision of an isolated antigen bindingprotein that binds HB-EGF, the antigen binding protein including (A) alight chain complementary determining region (CDRL) selected from thegroup consisting of (i) a CDRL3 selected from the group consisting ofSEQ ID NO:234-274, (ii) a CDRL3 that differs in amino acid sequence fromthe CDRL3 of (i) by an amino acid addition, deletion or substitution ofnot more than two amino acids; (iii) a CDRL3 amino acid sequenceselected from the group consisting of X₁QX₂X₃X₄X₅PX₆X₇ (SEQ ID NO:1046),wherein X₁ is selected from the group consisting of I and M, X₂ isselected from the group consisting of A, G and S, X₃ is selected fromthe group consisting of I and T, X₄ is selected from the groupconsisting of H and Q, X₅ is selected from the group consisting of F, Land W, X₆ is selected from the group consisting of C, I, H, L and T, X₇is selected from the group consisting of S and T; QQX₁X₂X₃X₄X₅IT (SEQ IDNO:1047), wherein X₁ is selected from the group consisting of I and S,X₂ is selected from the group consisting of F and Y, X₃ is selected fromthe group consisting of F, I, S and Y, X₄ is selected from the groupconsisting of A, S and T, X₅ is selected from the group consisting of Pand S; X₁X₂X₃X₄X₅X₆X₇X₈T (SEQ ID NO:1048), wherein X₁ is selected fromthe group consisting of L and Q, X₂ is selected from the groupconsisting of K, N and Q, X₃ is selected from the group consisting of A,H, S and Y, X₄ is selected from the group consisting of H, N and Y, X₅is selected from the group consisting of N, S and T, X₆ is selected fromthe group consisting of A, F, I, T, V and Y, X₇ is selected from thegroup consisting of P and no amino acid, X₈ is selected from the groupconsisting of F, L and P; QX₁X₂DX₃LPX₄X₅ (SEQ ID NO:1049), wherein X₁ isselected from the group consisting of H and Q, X₂ is selected from thegroup consisting of C and Y, X₃ is selected from the group consisting ofD, I, N, S and Y, X₄ is selected from the group consisting of F, I andL, X₅ is selected from the group consisting of A, S and T;QQX₁X₂X₃X₄PX₅X₆X₇ (SEQ ID NO:1050), wherein X₁ is selected from thegroup consisting of H and Y, X₂ is selected from the group consisting ofG and N, X₃ is selected from the group consisting of N and S, X₄ isselected from the group consisting of S and W, X₅ is selected from thegroup consisting of P and no amino acid, X₆ is selected from the groupconsisting of R and W, X₇ is selected from the group consisting of S andT; and X₁QYX₂X₃X₄X₅X₆X₇F (SEQ ID NO:1051), wherein X₁ is selected fromthe group consisting of H and Q, X₂ is selected from the groupconsisting of F and Y, X₃ is selected from the group consisting of G, Iand S, X₄ is selected from the group consisting of F, I and T, X₅ isselected from the group consisting of M, P, S and T, X₆ is selected fromthe group consisting of F, L, R and W, X₇ is selected from the groupconsisting of S and T; and/or (B) a heavy chain complementarydetermining region (CDRH) selected from the group consisting of (i) aCDRH3 selected from the group consisting of SEQ ID NOs:332-372, (ii) aCDRH3 that differs in amino acid sequence from the CDRH3 of (i) by anamino acid addition, deletion or substitution of not more than two aminoacids; and (iii) a CDRH3 amino acid sequence selected from the groupconsisting of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁DX₁₂ (SEQ ID NO:1065), wherein X₁is selected from the group consisting of E and S, X₂ is selected fromthe group consisting of D, G and no amino acid, X₃ is selected from thegroup consisting of D, N and no amino acid, X₄ is selected from thegroup consisting of G and no amino acid, X₅ is selected from the groupconsisting of G and no amino acid, X₆ is selected from the groupconsisting of W, Y and no amino acid, X₇ is selected from the groupconsisting of I, N and Y, X₈ is selected from the group consisting of Aand Y, X₉ is selected from the group consisting of G, V and Y, X₁₀ isselected from the group consisting of A, F and G, X₁₁ is selected fromthe group consisting of F, L and M, X₁₂ is selected from the groupconsisting of V and Y; QX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁YX₁₂X₁₃X₁₄DX₁₅ (SEQ IDNO:1066), wherein X₁ is selected from the group consisting of G and noamino acid, X₂ is selected from the group consisting of K, L and Y, X₃is selected from the group consisting of A, G and S, X₄ is selected fromthe group consisting of S, V and Y, X₅ is selected from the groupconsisting of A and G, X₆ is selected from the group consisting of G andno amino acid, X₇ is selected from the group consisting of T and noamino acid, X₈ is selected from the group consisting of S and no aminoacid, X₉ is selected from the group consisting of Y and no amino acid,X₁₀ is selected from the group consisting of W and Y, X₁₁ is selectedfrom the group consisting of G, S and Y, X₁₂ is selected from the groupconsisting of F and Y, X₁₃ is selected from the group consisting of Gand no amino acid, X₁₄ is selected from the group consisting of M and noamino acid, X₁₅ is selected from the group consisting of V and Y;X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO:1067), wherein X₁ isselected from the group consisting of D, G, L, S and no amino acid, X₂is selected from the group consisting of G, H, W, Y and no amino acid,X₃ is selected from the group consisting of A, F, W, Y and no aminoacid, X₄ is selected from the group consisting of D, G, Q, T and noamino acid, X₅ is selected from the group consisting of G, I, Q, S andno amino acid, X₆ is selected from the group consisting of A, D, X₈ isselected from the group consisting of D, Y and no amino acid, X₉ isselected from the group consisting of Y and no amino acid, X₁₀ isselected from the group consisting of A, E, N and Y, X₁₁ is selectedfrom the group consisting of G, P, T, V and Y, X₁₂ is se X₁₄ is selectedfrom the group consisting of C, H, V and Y;X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇DX₁₈ (SEQ ID NO:1068), whereinX₁ is selected from the group consisting of E, D and no amino acid, X₂is selected from the group consisting of G, R and no amino acid, X₃ isselected from the group consisting of I, V, Y and no amino acid, X₄ isselected from the group consisting of A, G, L and N, X₅ is selected fromthe group consisting of A, G, V and W, X₆ is selected from the groupconsisting of A, N, R and T, X₇ is selected from the group consisting ofG, N, P and no amino acid, X₈ is selected from the group consisting ofG, T and no amino acid, X₉ is selected from the group consisting of Aand no amino acid, X₁₀ is selected from the group consisting of D, E andno amino acid, X₁₁ is selected from the group consisting of S, Y and noamino acid, X₁₂ is selected from the group consisting of G, Y and noamino acid, X₁₃ is selected from the group consisting of N, Y and noamino acid, X₁₄ is selected from the group consisting of Y and no aminoacid, X₁₅ is selected from the group consisting of D, Y and no aminoacid, X₁₆ is selected from the group consisting of A, G and no aminoacid, X₁₇ is selected from the group consisting of F and M, X₁₈ isselected from the group consisting of I, V and Y;X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃ (SEQ IDNO:1069), wherein X₁ is selected from the group consisting of A, D, G, Sand T, X₂ is selected from the group consisting of A, E, G, L, N, R, Yand no amino acid, X₃ is selected from the group consisting of A, G, L,N, R, T, Y and no amino acid, X₄ is selected from the group consistingof D, G, R, S, V, Y and no amino acid, X₅ is selected from the groupconsisting of A, G, I, S, V, Y and no amino acid, X₆ is selected fromthe group consisting of F, G, L, R, V and no amino acid, X₇ is selectedfrom the group consisting of L, T, Y and no amino acid, X₈ is selectedfrom the group consisting of Y and no amino acid, X₉ is selected fromthe group consisting of Y and no amino acid, X₁₀ is selected from thegroup consisting of D and no amino acid, X₁₁ is selected from the groupconsisting of S and no amino acid, X₁₂ is selected from the groupconsisting of S and no amino acid, X₁₃ is selected from the groupconsisting of G and no amino acid, X₁₄ is selected from the groupconsisting of D, L, M, S, Y and no amino acid, X₁₅ is selected from thegroup consisting of H, I, P, V, W and no amino acid, X₁₆ is selectedfrom the group consisting of F, G, L, R, S, Y and no amino acid, X₁₇ isselected from the group consisting of D, F, V, W, Y and no amino acid,X₁₈ is selected from the group consisting of C, F, L, P, S and Y, X₁₉ isselected from the group consisting of D, F, G and Y, X₂₀ is selectedfrom the group consisting of A, C, G, P, R, V and Y, X₂₁ is selectedfrom the group consisting of F, L, M, S and no amino acid, X₂₂ isselected from the group consisting of A, D and no amino acid, X₂₃ isselected from the group consisting of I, L, V, Y and no amino acid;X₁YSSGWX₂X₃YGX₄X₅DX₆ (SEQ ID NO:1070), wherein X₁ is selected from thegroup consisting of M and V, X₂ is selected from the group consisting ofS and no amino acid, X₃ is selected from the group consisting of F andno amino acid, X₄ is selected from the group consisting of V and noamino acid, X₅ is selected from the group consisting of F and M, X₆ isselected from the group consisting of V and Y; and RX₁X₂X₃PFX₄Y (SEQ IDNO:1071), wherein X₁ is selected from the group consisting of G, H, L, Nand R, X₂ is selected from the group consisting of E, T and W, X₃ isselected from the group consisting of L, N, T and V, X₄ is selected fromthe group consisting of D and E.

In one embodiment, the isolated antigen binding protein furthercomprises (A) a CDRL selected from the group consisting of: (i) a CDRL1selected from the group consisting of SEQ ID NO:189-217; (ii) a CDRL1that differs in amino acid sequence from the CDRL1 of (i) by an aminoacid addition, deletion or substitution of not more than two aminoacids; (iii) a CDRL1 amino acid sequence selected from the groupconsisting of X₁SSQSLX₂X₃SDGX₄TYLX₅ (SEQ ID NO:1035), wherein X₁ isselected from the group consisting of K and R, X₂ is selected from thegroup consisting of L and V, X₃ is selected from the group consisting ofH and Y, X₄ is selected from the group consisting of K and N, X₅ isselected from the group consisting of N, S and Y; RASQX₁₁SX₂YLN (SEQ IDNO:1036), wherein X₁ is selected from the group consisting of R, S andT, X₂ is selected from the group consisting of R and S; RASQX₁₁X₂X₃X₄LX₅(SEQ ID NO:1037), wherein X₁ is selected from the group consisting of D,G, S and T, X₂ is selected from the group consisting of A, R and S, X₃is selected from the group consisting of H, I, N, R, S and T, X₄ isselected from the group consisting of D, W and Y, X₅ is selected fromthe group consisting of A, G and N; QASQDIX₁X₂X₃LN (SEQ ID NO:1038),wherein X₁ is selected from the group consisting of S and T, X₂ isselected from the group consisting of D and N, X₃ is selected from thegroup consisting of S and Y; RASQX₁VX₂X₃X₄X₅LA (SEQ ID NO:1039), whereinX₁ is selected from the group consisting of S and T, X₂ is selected fromthe group consisting of I and S, X₃ is selected from the groupconsisting of R and S, X₄ is selected from the group consisting of S, Nand no amino acid, X₅ is selected from the group consisting of Y and noamino acid; and KSSQX₁X₂LX₃X₄SNNKNYLX₅ (SEQ ID NO:1040), wherein X₁ isselected from the group consisting of N and S, X₂ is selected from thegroup consisting of I and V, X₃ is selected from the group consisting ofD and Y, X₄ is selected from the group consisting of N, R and S, X₅ isselected from the group consisting of A and V; or (iv) a CDRL2 selectedfrom the group consisting of SEQ ID NO:218-233; (v) a CDRH2 that differsin amino acid sequence from the CDRL2 of (iv) by an amino acid addition,deletion or substitution of not more than two amino acids; or (vi) aCDR12 amino acid sequence selected from the group consisting ofX₁X₂SNX₃X₄S (SEQ ID NO:1041), wherein X₁ is selected from the groupconsisting of E and K, X₂ is selected from the group consisting of I andV, X₃ is selected from the group consisting of R and W, X₄ is selectedfrom the group consisting of D and F; X₁X₂SX₃LQS (SEQ ID NO:1042),wherein X₁ is selected from the group consisting of A and T, X₂ isselected from the group consisting of A, E and V, X₃ is selected fromthe group consisting of S and T; X₁ASX₂LQS (SEQ ID NO:1043), wherein X₁is selected from the group consisting of A and V, X₂ is selected fromthe group consisting of S and T; DASX₁LET (SEQ ID NO:1044), wherein X₁is selected from the group consisting of I and N; GASSRAT (SEQ IDNO:223); and WASX₁RES (SEQ ID NO:1045), wherein X₁ is selected from thegroup consisting of A and T; or B) a CDRH selected from the groupconsisting of: (i) a CDRH1 selected from the group consisting of SEQ IDNO:275-299; (ii) a CDRH1 that differs in amino acid sequence from theCDRH1 of (i) by an amino acid addition, deletion or substitution of notmore than two amino acids; (iii) a CDRH1 amino acid sequence selectedfrom the group consisting of GYTX₁TX₂X₃X₄X₅X₆ (SEQ ID NO:1052), whereinX₁ is selected from the group consisting of F and L, X₂ is selected fromthe group consisting of E, G and S, X₃ is selected from the groupconsisting of H, L and Y, X₄ is selected from the group consisting of G,S and Y, X₅ is selected from the group consisting of I and M, X₆ isselected from the group consisting of H and S; GYX₁FTSYWIG (SEQ IDNO:1053), wherein X₁ is selected from the group consisting of R and S;GFTFX₁SX₂X₃MH (SEQ ID NO:1054), wherein X₁ is selected from the groupconsisting of R and S, X₂ is selected from the group consisting of H andY, X₃ is selected from the group consisting of D and G; GFX₁FSX₂YX₃MX₄(SEQ ID NO:1055), wherein X₁ is selected from the group consisting of Pand T, X₂ is selected from the group consisting of A, R and S, X₃ isselected from the group consisting of A and S, X₄ is selected from thegroup consisting of N and S; GX₁SX₂SX₃X₄X₅X₆X₇WX₈ (SEQ ID NO:1056),wherein X₁ is selected from the group consisting of D and G, X₂ isselected from the group consisting of F, I and V, X₃ is selected fromthe group consisting of R, S and no amino acid, X₄ is selected from thegroup consisting of G, Y and no amino acid, X₅ is selected from thegroup consisting of D, G, S and no amino acid, X₆ is selected from thegroup consisting of A, S and Y, X₇ is selected from the group consistingof A and Y, X₈ is selected from the group consisting of N and S;GFSLSNARMGVS (SEQ ID NO:279); and GFSLX₁TGGVGVG (SEQ ID NO:1057),wherein X₁ is selected from the group consisting of S and N; (iv) aCDRH2 selected from the group consisting of SEQ ID NO:300-331; (v) aCDRH2 that differs in amino acid sequence from the CDRH2 of (iv) by anamino acid addition, deletion or substitution of not more than two aminoacids; or (vi) a CDRH2 amino acid sequence selected from the groupconsisting of X₁X₂X₃X₄X₅X₆GX₇TX₈X₉X₁₀QKX₁₁X₁₂ (SEQ ID NO:1058), whereinX₁ is selected from the group consisting of S and W, X₂ is selected fromthe group consisting of F and I, X₃ is selected from the groupconsisting of D, N and S, X₄ is selected from the group consisting of Aand P, X₅ is selected from the group consisting of E, N and S, X₆ isselected from the group consisting of D, N and S, X₇ is selected fromthe group consisting of E, G and N, X₈ is selected from the groupconsisting of I and N, X₉ is selected from the group consisting of C, Hand Y, X₁₀ is selected from the group consisting of A and T, X₁₁ isselected from the group consisting of F and L, X₁₂ is selected from thegroup consisting of D and G; IIYPX₁DSDX₂RYSPSFQG (SEQ ID NO:1059),wherein X₁ is selected from the group consisting of D and G, X₂ isselected from the group consisting of A, I and T;X₁IX₂X₃DGSX₄X₅X₆YX₇DSVX₈G (SEQ ID NO:1060), wherein X₁ is selected fromthe group consisting of F and V, X₂ is selected from the groupconsisting of S and W, X₃ is selected from the group consisting of D, Sand Y, X₄ is selected from the group consisting of I, N and T, X₅ isselected from the group consisting of K and Q, X₆ is selected from thegroup consisting of N, R and Y, X₇ is selected from the group consistingof A, T and V, X₈ is selected from the group consisting of K and R;X₁ISX₂SX₃X₄X₅X₆YYADSVKG (SEQ ID NO:1061), wherein X₁ is selected fromthe group consisting of A, H and Y, X₂ is selected from the groupconsisting of G, R and S, X₃ is selected from the group consisting of Gand S, X₄ is selected from the group consisting of G, R and S, X₅ isselected from the group consisting of S, T and Y, X₆ is selected fromthe group consisting of I and T; X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁YX₁₂X₁₃SX₁₄KS(SEQ ID NO:1062), wherein X₁ is selected from the group consisting of E,R and Y, X₂ is selected from the group consisting of I and T, X₃ isselected from the group consisting of H, N and Y, X₄ is selected fromthe group consisting of C, H, S, T and Y, X₅ is selected from the groupconsisting of S and R, X₆ is selected from the group consisting of G andS, X₇ is selected from the group consisting of G, K, S and T, X₈ isselected from the group consisting of T and W, X₉ is selected from thegroup consisting of N and Y, X₁₀ is selected from the group consistingof N and no amino acid, X₁₁ is selected from the group consisting of Dand no amino acid, X₁₂ is selected from the group consisting of A and N,X₁₃ is selected from the group consisting of P and V, X₁₄ is selectedfrom the group consisting of L and V; X₁IFSNDEKSYSTSLKS (SEQ IDNO:1063), wherein X₁ is selected from the group consisting of H and LI;and LIYWNX₁X₂KRYSPSLX₃S (SEQ ID NO:1064), wherein X₁ is selected fromthe group consisting of D and V, X₂ is selected from the groupconsisting of D and E, X₃ is selected from the group consisting of K andR.

In some embodiments, at least two of, or all three of CDRL1, CDRL2, andCDRL3 sequences are derived from the same group A, B, C, D, E, or F ofconsensus sequences, and/or at least two, or all three of, CDRH1, CDRH2,and CDRH3 sequences are derived from the same group A, B, C, D, E, F, orG. In other cases CDRs from different consensus sequence groups aremixed and matched.

In yet another embodiment, the isolated antigen binding proteindescribed hereinabove comprises the first amino acid sequence and thesecond amino acid sequence, both sequences of which are covalentlybonded to each other. In a further embodiment, the first amino acidsequence of the isolated antigen binding protein includes the CDRL3 ofSEQ ID NO:234-274, CDRL2 of SEQ ID NO:218-233, and CDRL1 of SEQ IDNO:189-217. On the other hand, the second amino acid sequence of theisolated antigen binding protein comprises the CDRH3 of SEQ IDNO:332-372, CDRH2 of SEQ ID NO:300-331, and CDRH1 of SEQ ID NO:275-299.

In one aspect, the isolated antigen binding proteins provided herein canbe a monoclonal antibody, a polyclonal antibody, a recombinant antibody,a human antibody, a humanized antibody, a chimeric antibody, amultispecific antibody, or an antibody fragment thereof.

In another embodiment, the antibody fragment of the isolated antigenbinding proteins provided herein can be a Fab fragment, a Fab′ fragment,an F(ab′)₂ fragment, an Fv fragment, a diabody, or a single chainantibody molecule.

In a further embodiment, the isolated antigen binding protein providedherein is a human antibody and can be of the IgG1-, IgG2-IgG3- orIgG4-type.

In yet another aspect, the isolated antigen binding protein providedherein can be coupled to a labeling group, such as radioisotope,radionuclide, a fluorescent group, an enzymatic group, achemiluminescent group, a biotinyl group, or a predetermined polypeptidegroup, or an effector group, such as a radioisotope, a radionuclide, atoxin, a therapeutic group, or a chemotherapeutic group. Examples of atherapeutic or chemotherapeutic group are calicheamicin, auristatin-PE,geldanamycin, maytanasine, or derivatives thereof.

In yet other aspects, the invention includes antigen binding proteinscompeting with any of the above described antigen binding proteins.

As will be appreciated by those in the art, for any antigen bindingprotein with more than one CDR from the depicted sequences, anycombination of CDRs independently selected from the depicted sequencesis useful. Thus, antigen binding proteins with one, two, three, four,five or six of independently selected CDRs can be generated. However, aswill be appreciated by those in the art, specific embodiments generallyutilize combinations of CDRs that are non-repetitive, e.g., antigenbinding proteins are generally not made with two CDRH2 regions, etc.

Some of the antigen binding proteins provided are discussed in moredetail below.

1. Antigen Binding Proteins and Binding Epitopes

When an antigen binding protein is said to bind an epitope withinspecified residues of a polypeptide, such as HB-EGF, for example, whatis meant is that the antigen binding protein specifically binds to apolypeptide consisting of the specified residues (e.g., a specifiedsegment of HB-EGF). Such an antigen binding protein typically does notcontact every residue within HB-EGF. Nor does every single amino acidsubstitution or deletion within HB-EGF, or the extracellular domain ofHB-EGF, necessarily significantly affect binding affinity. Epitopespecificity of an antigen binding protein can be determined in varietyof ways. One approach, for example, involves testing a collection ofoverlapping peptides of about 15 amino acids spanning the sequence ofthe antigen and differing in increments of a small number of amino acids(e.g., three amino acids). The peptides are immobilized within the wellsof a microtiter dish. Immobilization can be effected by biotinylatingone terminus of the peptides. Optionally, different samples of the samepeptide can be biotinylated at the amino- and the carboxy-terminus andimmobilized in separate wells for purposes of comparison. This is usefulfor identifying end-specific antigen binding proteins. Optionally,additional peptides can be included by terminating at a particular aminoacid of interest. This approach is useful for identifying end-specificantigen binding proteins to internal fragments of HB-EGF. An antigenbinding protein or immunologically functional fragment is screened forspecific binding to each of the various peptides. The epitope is definedas occurring with a segment of amino acids that is common to allpeptides to which the antigen binding protein shows specific binding.Details regarding a specific approach for defining an epitope are setforth in Example 23.

As demonstrated in Example 23, the antigen binding proteins providedherein are capable of binding at least one IHGE-containing epitopeand/or an EGF-like domain of HB-EGF.

2. Competing Antigen Binding Proteins

In another aspect, antigen binding proteins are provided that competewith one of the exemplified antibodies or functional fragments bindingto the epitope described above for specific binding to HB-EGF. Suchantigen binding proteins may also bind to the same epitope as one of theherein exemplified antigen binding proteins, or an overlapping epitope.Antigen binding proteins and fragments that compete with or bind to thesame epitope as the exemplified antigen binding proteins are expected toshow similar functional properties. The exemplified antigen bindingproteins and fragments include those described above, including thosewith the heavy and light chains, variable region domains and CDRsincluded in FIGS. 1, 2, 3, 4, 6, and 7.

3. Human Antibodies and Humanization of Antibodies

In one embodiment, the HB-EGF antigen binding proteins are human orhumanized antibodies. Human antibodies avoid many of the problemsassociated with antibodies that possess murine or rat variable and/orconstant regions. The presence of such murine or rat derived proteinscan lead to the rapid clearance of the antibodies or can lead to thegeneration of an immune response against the antibody by a patient. Inorder to avoid the utilization of murine or rat derived antibodies,fully human antibodies have been generated through the introduction offunctional human antibody genetic loci into a rodent, other mammal oranimal so that the rodent, other mammal or animal produces fully humanantibodies.

One method for generating fully human antibodies is through the use ofXenoMouse® strains of mice that have been engineered to contain up tobut less than 1000 kb-sized germline configured fragments of the humanheavy chain locus and kappa light chain locus. See, Mendez et al., 1997,Nature Genetics 15:146-156, and Green and Jakobovits, 1998, J. Exp. Med.188:483-495. The XenoMouse® strains are available from Abgenix, Inc.(Fremont, Calif.).

The production of the XenoMouse® strains of mice is discussed anddelineated in U.S. patent application Ser. No. 07/466,008, filed Jan.12, 1990, Ser. No. 07/610,515, filed Nov. 8, 1990, Ser. No. 07/919,297,filed Jul. 24, 1992, Ser. No. 07/922,649, filed Jul. 30, 1992, Ser. No.08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848, filed Aug. 27,1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No. 08/376,279,filed Jan. 20, 1995, Ser. No. 08/430, 938, filed Apr. 27, 1995, Ser. No.08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun. 5, 1995,Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837, filed Jun.5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser. No. 08/486,857,filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995, Ser. No.08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct. 2, 1996,Ser. No. 08/759,620, filed Dec. 3, 1996, U.S. Publication 2003/0093820,filed Nov. 30, 2001 and U.S. Pat. Nos. 6,162,963, 6,150,584, 6,114,598,6,075,181, and 5,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068506 B2, and 3 068 507 B2. See, also European Patent No., EP 0 463 151B1, grant published Jun. 12, 1996, International Patent Application No.,WO 94/02602, published Feb. 3, 1994, International Patent ApplicationNo., WO 96/34096, published Oct. 31, 1996, WO 98/24893, published Jun.11, 1998, WO 00/76310, published Dec. 21, 2000. The disclosures of eachof the above-cited patents, applications, and references are herebyincorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, andusually a second constant region (preferably a gamma constant region)are formed into a construct for insertion into an animal. This approachis described in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat.Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfortand Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Bernset al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, filed Aug.29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279,filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No.07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec. 16,1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762,filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of whichare hereby incorporated by reference. See, also European Patent No. 0546 073 B1, International Patent Application Nos. WO 92/03918, WO92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No.5,981,175, the disclosures of which are hereby incorporated by referencein their entirety.

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See, European PatentApplication Nos. 773 288 and 843 961, the disclosures of which arehereby incorporated by reference. Additionally, KM™ mice, which are theresult of cross-breeding of Kirin's Tc mice with Medarex's minilocus(Humab) mice have been generated. These mice possess the human IgHtranschromosome of the Kirin mice and the kappa chain transgene of theGenpharm mice (Ishida et al., 2002, Cloning Stem Cells 4:91-102).

Human antibodies can also be derived by in vitro methods. Suitableexamples include but are not limited to phage display (CAT, Morphosys,Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon),Affimed) ribosome display (CAT), yeast display, and the like.

E. Preparation of Antibodies

Antibodies, as described herein, were prepared through the utilizationof the XenoMouse®XenoMouse technology, as described herein. Such miceare capable of producing human immunoglobulin molecules and antibodiesand are substantially deficient in the production of murineimmunoglobulin molecules and antibodies. Technologies utilized forachieving production of human antibodies are disclosed in the patents,applications, and references disclosed herein. In some embodiments,transgenic production of mice and human antibodies is performed asdisclosed in U.S. patent application Ser. No. 08/759,620, filed Dec. 3,1996 and International Patent Application Nos. WO 98/24893, publishedJun. 11, 1998 and WO 00/76310, published Dec. 21, 2000, the disclosuresof which are hereby incorporated by reference. See, also Mendez et al.,1997, Nature Genetics 15:146-156, the disclosure of which is herebyincorporated by reference.

Through the use of such technology, fully human monoclonal antibodies toa variety of antigens have been produced. Essentially, XenoMouse® linesof mice are immunized with an antigen of interest (e.g., HB-EGF),lymphatic cells (such as B-cells) are recovered from the hyper-immunizedmice, and the recovered lymphocytes are fused with a myeloid-type cellline to prepare immortal hybridoma cell lines. These hybridoma celllines are screened and selected to identify hybridoma cell lines thatproduced antibodies specific to the antigen of interest. Thesupernatants might also be screened for immunoreactivity againstfragments of HB-EGF to further map the different antibodies for bindingto domains of functional interest on HB-EGF. The antibodies may also bescreened for binding to other ligand of EGFR or its family members,other related human chemokines and against the rat, the mouse, andnon-human primate, such as cynomolgus monkey, orthologues of HB-EGF, thelast to determine species cross-reactivity. Provided herein are methodsfor the production of multiple hybridoma cell lines that produceantibodies specific to HB-EGF. Further, provided herein arecharacterization of the antibodies produced by such cell lines,including nucleotide and amino acid sequence analyses of the heavy andlight chains of such antibodies.

Alternatively, instead of being fused to myeloma cells to generatehybridomas, B cells can be directly assayed. For example, B cells can beisolated from hyperimmune XenoMouse® mice and allowed to proliferate anddifferentiate into antibody-secreting plasma cells. Antibodies from thecell supernatants are then screened by ELISA for reactivity against theHB-EGF immunogen. The supernatants might also be screened forimmunoreactivity against fragments of HB-EGF to further map thedifferent antibodies for binding to domains of functional interest onHB-EGF. The antibodies may also be screened for binding to other ligandsof EGF receptor, or its family members, other related human chemokinesand against the rat, the mouse, and non-human primate, such ascynomolgus monkey, orthologues of HB-EGF, the last to determine speciescross-reactivity. B cells from wells containing antibodies of interestmay be immortalized by various methods including fusion to makehybridomas either from individual or from pooled wells, or by infectionwith EBV or transfection by known immortalizing genes and then platingin suitable medium. Alternatively, single plasma cells secretingantibodies with the desired specificities are then isolated using anHB-EGF-specific hemolytic plaque assay (Babcook et al., 1996, Proc.Natl. Acad. Sci. USA 93:7843-48). Cells targeted for lysis arepreferably sheep red blood cells (SRBCs) coated with the HB-EGF antigen.

As discussed, supra, there are a number of isotypes of antibodiesincluding without limitation the following: human IgG1, IgG2, IgG3 andIgG4. It will be appreciated that antibodies that are generated need notinitially possess such an isotype but, rather the antibody as generatedcan possess any isotype and that the antibody can be isotype-switched byusing the molecularly cloned V region genes or cloned constant regiongenes or cDNAs in appropriate expression vectors using conventionalmolecular biological techniques that are well known in the art and thenexpressing the antibodies in host cells using techniques known in theart

In general, antibodies produced by the fused hybridomas were eitherhuman IgG2 heavy chains or human IgG4 heavy chains with fully humankappa chains. Antibodies can also be of other human isotypes, includingIgG1 or IgG3. The antibodies possessed high affinities, typicallypossessing a K_(D) of from about 10⁻⁶ through about 10⁻¹² M or below,when measured by solid phase and solution phase techniques. Antibodiespossessing a K_(D) of at least 10⁻⁹ M are preferred to inhibit theactivity of HB-EGF. Antibodies possessing a K_(D) of at least 10⁻¹⁹ Mare also preferred to inhibit the activity of HB-EGF. Antibodiespossessing a K_(D) of at least 10⁻¹¹ M are also preferred to inhibit theactivity of HB-EGF.

As will be appreciated, anti-HB-EGF antibodies can be expressed in celllines other than hybridoma cell lines. Sequences encoding particularantibodies can be used to transfect a suitable mammalian host cell.During construction of appropriate vectors for transfection andsubsequent expression of antibody, the antibody may be class-switchedfrom one isotype to another, e.g., IgG4 antibodies may be class-switchedto IgG2, by techniques known in the art. Transfection can be by anyknown method for introducing polynucleotides into a host cell,including, for example packaging the polynucleotide in a virus (or intoa viral vector) and transducing a host cell with the virus (or vector)or by transfection procedures known in the art, as exemplified by U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patentsare hereby incorporated herein by reference). The transformationprocedure used depends upon the host to be transformed. Methods forintroducing heterologous polynucleotides into mammalian cells are wellknown in the art and include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), human epithelial kidney 293 cells, and a number of othercell lines. Cell lines of particular preference are selected throughdetermining which cell lines have high expression levels and producehigh amounts of anti-HB-EGF antibodies.

Alternatively, these antibodies may be prepared from animals geneticallyengineered to make fully human antibodies or from an antibody displaylibrary made in bacteriophage, yeast, ribosome or E. coli. See, e.g.,Clackson et al., 1991, Nature 352:624-628, Marks et al., 1991, J. Mol.Biol. 222: 581-597, Feldhaus and Siegel, 2004, J. Immunol. Methods.290:69-80, Groves and Osbourn, 2005, Expert Opin Biol Ther. 5:125-135and Jostock and Dubel, 2005, Comb Chem High Throughput Screen.8:127-133.

Another aspect relates to an isolated nucleic acid molecule encoding anHB-EGF antigen binding protein such as an antibody. Within the contextherein, the term “isolated nucleic acid molecule”, as used herein, meansa polynucleotide of genomic, cDNA, or synthetic origin or somecombination thereof, which by virtue of its origin, the “isolatednucleic acid molecule” (1) is not associated with all or a portion of apolynucleotide in which the “isolated polynucleotide” is found innature, (2) is operably linked to a polynucleotide which it is notlinked to in nature, or (3) does not occur in nature as part of a largersequence. Further, the term “nucleic acid molecule”, as referred toherein, means a polymeric form of nucleotides of at least 10 bases inlength, either ribonucleotides or deoxynucleotides or a modified form ofeither type of nucleotide, such as nucleotides with modified orsubstituted sugar groups and the like. The term also includes single anddouble stranded forms of DNA. Exemplatory nucleic acids encoding antigenbinding proteins or portions thereof are described in more detail,infra.

In a one embodiment, a nucleic acid molecule is operably linked to acontrol sequence. The term “control sequence”, as used herein, refers topolynucleotide sequences that are necessary to effect the expression andprocessing of coding sequences to which they are ligated. The nature ofsuch control sequences differs depending upon the host organism. Inprokaryotes, such control sequences generally include promoters,ribosomal binding sites, and transcription termination sequences. Ineukaryotes, generally, such control sequences include promoters andtranscription termination sequences. The term “control sequence” isintended to include, at a minimum, all components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Furthermore, the term “operably linked”,as used herein, refers to positions of components so described which arein a relationship permitting them to function in their intended manner.Moreover, as provided herein, an expression control sequence operablylinked to a coding sequence is ligated in such a way that expression ofthe coding sequence is achieved under conditions compatible with theexpression control sequence.

A further aspect is a vector comprising a nucleic acid molecule thatencodes an HB-EGF antigen binding protein provided herein. The nucleicacid molecule can be operably linked to a control sequence. Furthermore,the vector may additionally contain a replication origin or a selectionmarker gene. Examples of vectors that may be used are e.g., plasmids,cosmids, phages, viruses, etc.

F. Antigen Binding Proteins Based on Basic Antibody Structure

As discussed, supra, the basic antibody structural unit is known tocomprise a tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region responsible fordimerization effector function, circulating half-life and otherfunctions. Human light chains are classified as kappa and lambda lightchains. Heavy chains are classified as mu, delta, gamma, alpha, orepsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, andIgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See, generally, Fundamental Immunology Ch. 7 (Paul, W.,ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in itsentirety for all purposes). The variable regions of each light/heavychain pair form the antigen binding site of the antibody.

Thus, an intact IgG antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame.

The variable regions of the chains all exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehyper variable regions, also called complementarity determining regionsor CDRs. The CDRs from the two variable regions of each pair are alignedby the framework regions, enabling binding to a specific epitope on theantigen. From N-terminal to C-terminal, the variable regions of bothlight and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3,CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk, 1897, J. Mol. Biol. 196:901-917;Chothia et al., 1989, Nature 342:878-883.

Thus, the antibodies provided herein may include at least one variableregion polypeptide chain of the formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4,wherein FR1 is a first human framework region, CDR1 is a firstcomplementarity determining region, FR2 is a second human frameworkregion, CDR2 is a second complementarity determining region, FR3 is athird human framework region, CDR3 is a third complementaritydetermining region, and FR4 is a fourth human framework region. The CDR3is generally the most diverse region of the antibody variable region.

In some embodiments, the FR1 region includes but is not limited to anyone of amino acid sequences SEQ ID NOs:373-393 and/or 453-469; the CDR1region includes but is not limited to any one of amino acid sequencesSEQ ID NOs:189-217 and/or 275-299; the FR2 region includes but is notlimited to any one of amino acid sequences SEQ ID NOs:394-414 and/or470-481; the CDR2 region includes but is not limited to any one of aminoacid sequences SEQ ID NOs:218-233 and/or 300-331; the FR3 regionincludes but is not limited to any one of amino acid sequences SEQ IDNOs:415-440 and/or 482-511; the CDR3 region includes but is not limitedto any one of amino acid sequences SEQ ID NOs:234-274 and/or 332-372;and the FR4 region includes but is not limited to any one of amino acidsequences SEQ ID NOs:441-452 and/or 512-517. Thus, in some embodiments,the human antibodies provided herein have one or more of the amino acidsequences provided herein.

It is to be understood, that the amino acid sequence of the antibodiesprovided herein is not limited to the twenty conventional amino acids(See, Immunology—A Synthesis (2^(nd) Edition, E. S. Golub and D. R.Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which isincorporated herein by reference). For example, the amino acids mayinclude stereoisomers (e.g., D-amino acids) of the twenty conventionalamino acids, unnatural amino acids such as α-,α-disubstituted aminoacids, N-alkyl amino acids, lactic acid, and other unconventional aminoacids. Examples of unconventional amino acids, which may also besuitable components for the antibody provided, include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, α-N-methylarginine, and othersimilar amino acids and imino acids, e.g., 4-hydroxyproline.

Furthermore, minor variations in the amino acid sequences shown in SEQID NOs:1-517 and 1035-1071 are contemplated as being encompassed,providing that the variations in the amino acid sequence maintain atleast 75%, more preferably at least 80%, 90%, 95%, and most preferably99% of the sequences shown in SEQ ID NOs:1-517 and 1035-1071. Preferredvariations in the amino acid sequences shown in SEQ ID Nos:1-517 and1035-1071, i.e., deletions, insertions and/or replacements of at leastone amino acid, occur near boundaries of functional domains. Structuraland functional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Computerized comparison methods can be used to identifysequence motifs or predicted protein conformation domains that occur inother antibodies of known structure and/or function. Methods to identifyprotein sequences that fold into a known three-dimensional structure areknown. See, e.g., Bowie et al., 1991, Science 253:164; Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al., 1991, Nature 354:105, which are all incorporated hereinby reference. Thus, those of skill in the art can recognize sequencemotifs and structural conformations that may be used to definestructural and functional domains.

Especially preferred variations in the amino acid sequences shown in SEQID NOs:1-517 and 1035-1071 are those that lead to a reducedsusceptibility to proteolysis or oxidation, alter glycosylation patternsor alter binding affinities or confer or modify other physicochemical orfunctional properties of the antibody. In particular, conservative aminoacid replacements are contemplated. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Preferred amino acid families are the following: acidicfamily=aspartate, glutamate; basic family=lysine, arginine, histidine;non-polar family=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and uncharged polarfamily=glycine, asparagine, glutamine, cysteine, serine, threonine,tyrosine. More preferred families are: aliphatic-hydroxy family=serineand threonine; amide-containing family=asparagine and glutamine;aliphatic family=alanine, valine, leucine and isoleucine; and aromaticfamily=phenylalanine, tryptophan, and tyrosine. For example, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting antibody, especially if the replacement doesnot involve an amino acid within a framework site. However, all otherpossible amino acid replacements are also encompassed. Whether an aminoacid change results in a functional antibody, i.e., in a antibody thatbinds to HB-EGF and reduces, neutralizes or substantially inhibits thefunction of HB-EGF, can readily be determined by assaying the specificactivity of the resulting antibody in ELISA or FACS for binding toHB-EGF or in vitro or in vivo functional assay.

A reduction, neutralization or substantially inhibition of HB-EGFmediated signal transduction may be caused by influencing, e.g.,decreasing or inhibiting, the binding of HB-EGF to its receptor, e.g.,to the EGFR or HER4.

The term “antibody” or “anti-HB-EGF antibody”, as used herein, means amonoclonal antibody, a polyclonal antibody, a recombinant antibody, ahumanized antibody (Jones et al., 1986, Nature 321:522-525; Riechmann etal., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol.2: 593-596), a chimeric antibody (Morrison et al., 1984, Proc. Natl.Acad. Sci. U.S.A. 81: 6851-6855), a multispecific antibody (e.g., abispecific antibody) formed from at least two antibodies, or an antibodyfragment thereof. The term “antibody fragment” comprises any portion ofthe afore-mentioned antibodies, preferably at least one of their antigenbinding or variable regions. Examples of antibody fragments include Fabfragments, Fab′ fragments, F(ab′)₂ fragments, Fv fragments, diabodies(Hollinger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 6444-6448),single chain antibody molecules (Plückthun in: The Pharmacology ofMonoclonal Antibodies 113, Rosenburg and Moore, EDS, Springer Verlag,N.Y. (1994), 269-315) and other fragments as long as they exhibit thedesired capability of binding to HB-EGF.

In addition, the term “antibody” or “anti-HB-EGF antibody”, as usedherein, may include antibody-like molecules that contain engineeredsub-domains of antibodies or naturally occurring antibody variants.These antibody-like molecules may be single-domain antibodies such asVH-only or VL-only domains derived either from natural sources such ascamelids (Muyldermans et al., 2001, Reviews in Molecular Biotechnology74, 277-302) or through in vitro display of libraries from humans,camelids or other species (Holt et al., 2003, Trends Biotechnol.21:484-90).

A “Fv fragment” is the minimum antibody fragment that contains acomplete antigen-recognition and -binding site. This region consists ofa dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. It is in this configuration that the threeCDR's of each variable domain interact to define an antigen-binding siteon the surface of the V_(H)-V_(L) dimer. Collectively, the six CDR'sconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDR'sspecific for an antigen) has the ability to recognize and bind theantigen, although usually at a lower affinity than the entire bindingsite. The “Fab fragment” also contains the constant domain of the lightchain and the first constant domain (C_(H)1) of the heavy chain. The“Fab fragment” differs from the “Fab′ fragment” by the addition of a fewresidues at the carboxy terminus of the heavy chain C_(H)1 domainincluding one or more cysteines from the antibody hinge region. The“F(ab′)₂ fragment” originally is produced as a pair of “Fab′ fragments”which have hinge cysteines between them. Methods of preparing suchantibody fragments, such as papain or pepsin digestion, are known tothose skilled in the art.

An antibody provided herein may fix complement (CDC) or activateantibody-dependent cellular cytotoxicity (ADCC), especially an IgG1antibody, IgG1 variant class-switched from IgG2 or IgG4 or anotherisotype of human or mammalian origin, by molecular biology or generatedde novo from human IgG1 producing mice. Other methods may also be used.An antibody provided herein may sometimes be coupled to a labellinggroup or an effector group, e.g., a toxin, chemotherapeutic agent,reporter molecule or imaging agent.

G. Further Types of HB-EGF Binding Proteins

In another aspect, the HB-EGF antigen binding protein as provided hereinis a scaffold protein having an antibody like binding activity, i.e.,binds to HB-EGF.

Within the context of the present invention, the term “scaffoldprotein”, as used herein, means a polypeptidic framework with a hightolerance of its fold for modifications such as multiple insertions,deletions or substitutions. This intrinsic conformational stabilityenables the directed randomization and drastic changes within a definedregion of the protein. Thus, it acquires certain novel properties,whereas its overall structural integrity and original physicochemicalbehaviour remains conserved. This de novo adopted property mostly, butnot exclusively, comprises the binding specificity for a pre-definedtarget molecule.

Currently, a broad variety of scaffold proteins are in use that imitatethe binding principle of a conventional antibody to different degrees(Hey et al., 2005, Trends in Biotechnol. 23:514-22). Examples ofscaffold proteins that can be used in accordance with the presentinvention can be subdivided into different groups. Antibody relatedscaffolds, which are defined as derivatives of antibodies that areeither naturally smaller in size and simpler in structure, or have beenengineered in this way. This group includes for example so calledNanobodies (reviewed in Hey et al., Trends in Biotechnol. 2005; 23 (10);514-522), domain antibodies (Holt et al., 2003, Trends in Biotechnol.21:484-489) or shark antigen reactive proteins (Holt et al., Trends inBiotechnol. 2003; 21; 484-489). A second group of scaffold proteins arerigid protein folds which tolerate the insertion or randomization ofsingle loop peptides like the Kunitz-type domain (Dennis et al., 1995;J. Biol. Cell. 270:25411-25417), huma transferrin (Ali et al., 1999; J.Biol. Cell. 274:24066-24074) or cystein-knot structural motives(Christmann et al., 1999, Protein Eng. 12:797-806). Proteins that sharethe principle of multiple hypervariable loops on a rigid conservedframework in analogy to antibodies are represented by human CTLA-4(Hufton et al., 2000 FEBS Lett. 475:225-231), human fibronectin type IIIdomains (Koide et al., 1998; J. Mol. Biol. 284:1141-1151), C-type lectinlike domains or lipocalins (Skerra, 2001, J. Biotechnol. 74:257-275).Scaffolds with the binding specificity accomplished through aminoacidresidues which are positioned partially or completely within the rigidsecondary structure of the protein are for example ankyrin repeatproteins (Binz, 2003, J. Mol. Biol. 332:489-503), the Z domain ofproteinA (Nord et al., 1995, Protein Eng. 8:601-608) or γ-crystalline(Fiedler and Rudolph, 2001, International Patent Application WO01/04144). Scaffold proteins and peptides and applications thereof arereviewed in Hey et al., 2005, Trends in Biotechnol. 23:514-522; Binz etal., 2005, Nature Biotechnol. 23:1257-1268) and Holliger et al., 2005,Nature Biotechnol. 23:11261136.

Engineering of a scaffold protein can be regarded as grafting orintegrating an affinity function onto or into the structural frameworkof a polypeptidic framework with a high tolerance of its fold formodifications. Affinity function means a protein binding affinityaccording to the present invention. A scaffold can be structurallyseparable from the amino acid sequences conferring binding specificity.In general, proteins appearing suitable for the development of suchartificial affinity reagents may be obtained by rational, or mostcommonly, combinatorial protein engineering techniques such as panningagainst an antigen, e.g, HB-EGF, either purified protein or proteindisplayed on the cell surface, for binding agents in an artificialscaffold library displayed in vitro, using skills which are known in theart (Skerra, 2000, J. Mol. Recog. July-August; 13(4):167-87; Binz andPlückthun, 2005, August; 16(4):459-69). In addition, a scaffold proteinhaving an antibody like binding activity can be derived from an acceptorpolypeptide, e.g one of the foregoing proteins, containing the scaffolddomain, which can be grafted with binding domains of a donor polypeptideto confer the binding specificity of the donor polypeptide onto thescaffold domain containing the acceptor polypeptide. Said insertedbinding domains may be, for example, one or more of the complementaritydetermining region (CDR) of an antibody, in particular an HB-EGFantibody. Preferably the CDR is a CDR3. Insertion can be accomplished byvarious methods known to those skilled in the art including, forexample, polypeptide synthesis, nucleic acid synthesis of an encodingamino acid as well by various forms of recombinant methods well known tothose skilled in the art.

H. HB-EGF Antigen Binding Protein Conjugates

In another embodiment, an HB-EGF antigen binding protein, e.g., anantibody provided herein is coupled to a labelling group. Such alabelled antigen binding protein is particularly suitable for diagnosticapplications. As used herein, the term “labelling group” refers to adetectable marker, e.g., a radiolabelled amino acid or biotinyl moietythat can be detected by marked avidin (e.g., streptavidin bound to afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Various methods for labelling polypeptides andglycoproteins, such as antibodies, are known in the art and may be used.Examples of suitable labelling groups include, but are not limited to,the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S,⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups e. FITC, rhodamine,lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentgroups, biotinyl groups, or predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In certain respects, it may be desirable that the labellinggroups are attached by spacer arms of various lengths to reducepotential steric hindrance.

Alternatively, an HB-EGF antigen binding protein provided herein, suchas an antibody, may be coupled to an effector group. Such aneffector-modified antigen binding protein is especially suitable fortherapeutic applications. As used herein, the term “effector group”refers to a cytotoxic group such as a radioisotope or radionuclide, atoxin, a therapeutic group or other effector group known in the art.Examples for suitable effector groups are radioisotopes or radionuclides(e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), calicheamicin,dolastatin analogs such as auristatins, and chemotherapeutic agents suchas geldanamycin and maytansine derivates, including DM1. In certainrespects, it may be desirable that the effector groups are attached byspacer arms of various lengths to reduce potential steric hindrance.

Also as described herein, many of the highly useful HB-EGF antigenbinding protein, e.g., antibody preparations provided herein recognizeepitopes within the EGF-like domain of HB-EGF, which includes residues106-149 of the protein, for example, with the following sequence:

PCLRKYKDFCIHGECKYVKELRAPSCICHPGY HGERCHGLSLP (SEQ ID NO:1076). In someembodiments, the epidermal pgrowth factor epitope recognized by theantigen binding proteins provided herein includes amino acid sequenceIHGE. Accordingly, antibody preparations are provided that bind to andrecognize an IHGE-containing epitope and/or an EGF-like domain ofHB-EGF, for example, SEQ ID NO:1076.

Anti-HB-EGF antigen binding proteins are useful in the detection ofHB-EGF in patient samples and accordingly are useful as diagnostics fordisease states as described herein. In addition, based on their abilityto significantly inhibit HB-EGF and/or EGF receptor activity (asdemonstrated in the Examples below), HB-EGF antigen binding proteinshave therapeutic effects in treating symptoms and conditions resultingfrom HB-EGF expression and/or HB-EGF activity. In specific embodiments,the antigen binding proteins and methods herein relate to the treatmentof symptoms resulting from HB-EGF-associated diseases, HER4-associateddiseases or EGF receptor-associated disease states, for example,cancerous conditions. Further embodiments involve using the antigenbinding proteins and methods described herein to treat undesiredangiogenesis, neoplastic diseases, such as, melanoma, non-small celllung cancer, glioma, hepatocellular (liver) carcinoma, thyroid tumor,gastric (stomach) cancer, prostrate cancer, breast cancer, ovariancancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer,kidney cancer, colon cancer, and pancreatic cancer.

I. Nucleic Acids Encoding HB-EGF Antigen Binding Proteins

Nucleic acids that encode for the antigen binding proteins describedherein, or portions thereof, are also provided, including nucleic acidsencoding one or both chains of an antibody, or a fragment, derivative,mutein, or variant thereof, polynucleotides encoding heavy chainvariable regions or only CDRs, polynucleotides sufficient for use ashybridization probes, PCR primers or sequencing primers for identifying,analyzing, mutating or amplifying a polynucleotide encoding apolypeptide, anti-sense nucleic acids for inhibiting expression of apolynucleotide, and complementary sequences of the foregoing. Thenucleic acids can be any length. They can be, for example, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides inlength, and/or can comprise one or more additional sequences, forexample, regulatory sequences and/or be part of a larger nucleic acid,for example, a vector. The nucleic acids can be single-stranded ordouble-stranded and can comprise RNA and/or DNA nucleotides, andartificial variants thereof (e.g., peptide nucleic acids). FIGS. 15A-15Vdepict the nucleotide sequences for the various light chains of theantigen binding proteins. FIGS. 16A-16AC deptice the nucleotidesequences for the various heavy chains of the antigen binding proteins.FIGS. 13A-13M depict the nucleotide sequences of various light chainvariable regions of the antigen binding proteins. FIGS. 14A-14L depictthe nucleotide sequences of various heavy chain variable regions of theantigen binding proteins. FIGS. 18A-18F depict the nucleotide sequencesfor various CDR regions of the light chain variable regions of theantigen binding proteins. FIGS. 19A-19G depict the nucleotide sequencesfor various CDR regions of the heavy chain variable regions of theantigen binding proteins. FIGS. 20A-20K depict the nucleotide sequencesfor various FR regions of the light chain variable regions of theantigen binding proteins. FIGS. 21A-21K depict the nucleotide sequencesfor various FR regions of the heavy chain, variable regions of theantigen binding proteins. FIG. 17A depicts the nucleotide sequence ofthe light chain constant region of the antigen binding proteins.Finally, FIG. 17B depicts the nucleotide sequence of the heavy chainconstant region of the antigen binding proteins.

Nucleic acids encoding certain antigen binding proteins, or portionsthereof (e.g., full length antibody, heavy or light chain variabledomain or CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3) may be isolatedfrom B-cells of mice that have been immunized with HB-EGF or animmunogenic fragment thereof. The nucleic acid may be isolated byconventional procedures such as polymerase chain reaction (PCR). Phagedisplay is another example of a known technique whereby derivatives ofantibodies and other antigen binding proteins may be prepared. In oneapproach, polypeptides that are components of an antigen binding proteinof interest are expressed in any suitable recombinant expression system,and the expressed polypeptides are allowed to assemble to form antigenbinding protein molecules.

An aspect further provides nucleic acids that hybridize to other nucleicacids (e.g., nucleic acids comprising a nucleotide sequence depicted inFIGS. 13 through 21) under particular hybridization conditions. Methodsfor hybridizing nucleic acids are well-known in the art. See, e.g.,Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. As defined herein, a moderately stringent hybridizationcondition uses a prewashing solution containing 5× sodiumchloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization buffer of about 50% formamide, 6×SSC, and a hybridizationtemperature of 55° C. (or other similar hybridization solutions, such asone containing about 50% formamide, with a hybridization temperature of42° C.), and washing conditions of 60° C., in 0.5×SSC, 0.1% SDS. Astringent hybridization condition hybridizes in 6×SSC at 45° C.,followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C.Furthermore, one of skill in the art can manipulate the hybridizationand/or washing conditions to increase or decrease the stringency ofhybridization such that nucleic acids comprising nucleotide sequencesthat are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%identical to each other typically remain hybridized to each other.

The basic parameters affecting the choice of hybridization conditionsand guidance for devising suitable conditions are set forth by, forexample, Sambrook, Fritsch, and Maniatis (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., supra and Current Protocols in Molecular Biology, 1995,Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4), and can be readily determined by those having ordinary skillin the art based on, e.g., the length and/or base composition of thenucleic acid.

Changes can be introduced by mutation into a nucleic acid, therebyleading to changes in the amino acid sequence of a polypeptide (e.g., anantibody or antibody derivative) that it encodes. Mutations can beintroduced using any technique known in the art. In one embodiment, oneor more particular amino acid residues are changed using, for example, asite-directed mutagenesis protocol. In another embodiment, one or morerandomly selected residues is changed using, for example, a randommutagenesis protocol. However it is made, a mutant polypeptide can beexpressed and screened for a desired property.

Mutations can be introduced into a nucleic acid without significantlyaltering the biological activity of a polypeptide that it encodes. Forexample, one can make nucleotide substitutions leading to amino acidsubstitutions at non-essential amino acid residues. Alternatively, oneor more mutations can be introduced into a nucleic acid that selectivelychanges the biological activity of a polypeptide that it encodes. Forexample, the mutation can quantitatively or qualitatively change thebiological activity. Examples of quantitative changes includeincreasing, reducing or eliminating the activity. Examples ofqualitative changes include changing the antigen specificity of anantibody.

Another aspect provides nucleic acid molecules that are suitable for useas primers or hybridization probes for the detection of nucleic acidsequences. A nucleic acid molecule can comprise only a portion of anucleic acid sequence encoding a full-length polypeptide, for example, afragment that can be used as a probe or primer or a fragment encoding anactive portion (e.g., a HB-EGF binding portion) of a polypeptide.

Probes based on the sequence of a nucleic acid can be used to detect thenucleic acid or similar nucleic acids, for example, transcripts encodinga polypeptide. The probe can comprise a label group, e.g., aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used to identify a cell that expresses thepolypeptide.

Another aspect provides vectors comprising a nucleic acid encoding apolypeptide or a portion thereof (e.g., a fragment containing one ormore CDRs or one or more variable region domains). Examples of vectorsinclude, but are not limited to, plasmids, viral vectors, non-episomalmammalian vectors and expression vectors, for example, recombinantexpression vectors. The recombinant expression vectors can comprise anucleic acid in a form suitable for expression of the nucleic acid in ahost cell. The recombinant expression vectors include one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, which is operably linked to the nucleic acid sequence tobe expressed. Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcells (e.g., SV40 early gene enhancer. Rous sarcoma virus promoter andcytomegalovirus promoter), those that direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences, see, Voss et al., 1986 Trends Biochem. Sci.11:287, Maniatis et al., 1987, Science 236:1237, incorporated byreference herein in their entireties), and those that direct inducibleexpression of a nucleotide sequence in response to particular treatmentor condition (e.g., the metallothionin promoter in mammalian cells andthe tet-responsive and/or streptomycin responsive promoter in bothprokaryotic and eukaryotic systems (see, id.). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. The expression vectorscan be introduced into host cells to thereby produce proteins orpeptides, including fusion proteins or peptides, encoded by nucleicacids as described herein.

Another aspect provides host cells into which a recombinant expressionvector has been introduced. A host cell can be any prokaryotic cell (forexample, E. coli) or eukaryotic cell (for example, yeast, insect, ormammalian cells (e.g., CHO cells)). Vector DNA can be introduced intoprokaryotic or eukaryotic cells via conventional transformation ortransfection techniques. For stable transfection of mammalian cells, itis known that, depending upon the expression vector and transfectiontechnique used, only a small fraction of cells may integrate the foreignDNA into their genome. In order to identify and select these integrants,a gene that encodes a selectable marker (e.g., for resistance toantibiotics) is generally introduced into the host cells along with thegene of interest. Preferred selectable markers include those whichconfer resistance to drugs, such as G418, hygromycin and methotrexate.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die), amongother methods.

J. Use of HB-EGF Antigen Binding Proteins for Diagnostic and TherapeuticPurposes

1. Indications

The HB-EGF antigen binding proteins as described herein can be used todetect or treat a number of diseases and disorders, including thoseinvolving excessive cellular proliferation, undesirable cellularmigration and/or aberrant angiogenesis. For example, these HB-EGFantigen binding proteins can inhibit HB-EGF-induced EGFR and/or HER4tyrosine phosphorylation in cancerous cells (FIGS. 22-29). Suchinhibition can interrupt the cascade of the signaling events that drivescell proliferation and migration, and angiogenesis. Further, the HB-EGFantigen binding proteins can interfere with the transactivation of theEGFR. In addition, these antigen binding proteins inhibit basal HUVECcell proliferation (FIG. 32B), endothelial cell tube formation (FIG. 33)and HB-EGF-induced vessel formation in a matrigel plug assay in vivo(FIG. 36). These results indicate that the HB-EGF antigen bindingproteins as described herein inhibit angiogenesis in vitro and in vivo.Moreover, these antigen binding proteins also inhibit anchorageindependent cell growth (FIG. 34 and FIG. 35) and xenograft tumor growthin mice (FIG. 37 and FIG. 38). Significantly, the HB-EGF antigen bindingproteins also inhibit migration of MCF-7 and MDA-MB231 cancer cells(see, FIGS. 27 and 27). Thus, the HB-EGF antigen binding proteins asdescribed herein attack several steps in the development of tumors andother cancerous conditions, including signaling events that control cellproliferation, angiogenesis and cell migration associated with thespread and development of metastatic cancer. Such multi-facetedintervention is highly beneficial for controlling and inhibiting theprocess by which cancer develops. Furthermore, cancers at any stage ofprogression (e.g., primary, metastatic and recurrent cancers) can betreated.

In addition to the HB-EGF antigen binding proteins' use in detecting andtreating cancers in various stages of progression, these antigen bindingproteins may also be useful for detecting a number of different types ofcancer. For example, HB-EGF is expressed at very low levels in normalbreast and pancreatic tissues. However, HB-EGF is expressed at highlevels in about 55% pancreatic cancer cells and about 70% of breastcancer cells. Furthermore, as described in the Examples, HB-EGFexpression was detected in various cancer cell lines, indicating thatthe HB-EGF antigen binding proteins described herein can be used fordetection of a variety of cancer types.

For example, cancers that can be detected or treated by the claimedantigen binding proteins include solid mammalian tumors as well ashematological malignancies. Solid mammalian tumors include cancers inchildren such as, for example, germ cell tumors, soft tissue sarcomas,primary brain tumors, neuroblastoma, nephroblastoma and carcinoma, inparticular squamous carcinoma and epithelial carcinoma. Solid mammaliantumors may also include adult cancers such as, for example, tumors ofunknown origin, primary brain cancer in adults, tumors of the pituitarygland, lip, oral cavity, Nasopharynx, larynx, maxillary sinus, Ethmoidsinus, salivary glands, thyroid gland (including para thyroid glands andcarcinoid), esophagus, stomach, pancreas, small intestine, colon,rectum, anal canal, liver, gallbladder, extra hepatic bile ducts,ampulla of vater, carcinoid, endocrine tumors of gastro-entero-hepaticsystem, pheochromocytoma and paraganglioma, adrenal glands, lung,pleura, mediastinum, thymus, tumors of bone and soft tissue, skin tumorsof lip, eyelid, external ear, other unspecified parts of the face, scalpand neck, trunk, upper limpb and shoulder, lower limb and hip, vulva,penis, scrotum, breast tumors, gynecological tumors of vulva, vagina,cervix uteri, corpus uteri, ovary, fallopian tube, gestational andtrophoblastic tumors, penis, prostate, testis, kidney, renal pelvis andureter, urinary bladder, urethra, ophthalmic tumors of eyelid,conjunctiva, uvea, retina, orbit and lacrimal gland. Hematologicalmalignancies include childhood, for example, leukemia and lymphomas,acute and chronic leukemia (AML, ANLL, ALL, CML, MDS), Hodgkin'sdisease, B-Cell, T-Cell, large cell, follicular, indolent/low grade,aggressive/high grade lymphomas of lymphocytic and cutaneous origin,plasma cell neoplasm and cancers associated with AIDS.

In addition, the HB-EGF antigen binding proteins described herein mayalso be used to detect or treat cancerous conditions or neoplasiadisorders, which include, for example, adenoma, tubulovillous adenoma,villous adenoma, angiofibroma, atypical proliferating mucinousneoplasias, Brenner tumor, carcinoid, cavernous hemangioma, cellularleiomyoma, chorangioma, congenital mesoblastic nephroma, mucinouscystadenoma, serous cystadenoma, dermoid, desmoid, fibroadenoma,fibroma, fibrothecoma, follicular adenoma, ganglioneuroma, giant celltumor, granular cell tumor, granulosa cell tumor, hemangioma,intraductal papilloma, islet cell tumor, leiomyoma, lipoma, luteoma,meningioma, mole, myelolipoma, myxoma, neurofibroma, nevus,osteochondroma, pheochromocytoma, polyposis, schwannoma, serouscystadenoma, struma ovarii, synovial chrondromatosis, benign thymoma.

Further examples of the types of cancers that can be detected or treatedwith the HB-EGF proteins as described herein may be found, for example,from the American Cancer Society (www.cancer.org), or from Wilson et al.(1991) Harrison's Principles of Internal Medicine, 12^(th) Edition,McGraw-Hill, Inc.

Therefore, the HB-EGF antigen binding proteins as described herein canbe used to treat and/or prevent cancer, cancerous conditions, tumorgrowth, metastasis of cancer cells, angiogenic processes and/orneoplastic disorders. Thus, these antigen binding proteins provide amethod of treating or preventing cancer in a subject that involvesadministering to the subject an effective amount of a compositioncomprising one of the human and/or monoclonal HB-EGF antigen bindingprotein preparations as described herein, or a combination thereof.

A high proportion of solid tumor diseases are often characterized bytumor angiogenesis, the excessive growth of (abnormal) vessels in thetumor tissue mediated by growth factors (i.e., VEGF) and other factors(i.e., HB-EGF). Targeting HB-EGF through a HB-EGF-specific antigenbinding protein could prevent the formation of new vessels and thereforelimit the expansion of existing tumors and the development of new tumors(i.e., metastases).

Besides its role as a mitogenic and pro-invasive ligand several studieshave substantiated the picture of HB-EGF as an important regulator ofangiogenic processes in cancer. As illustrated in the Examples, afunction of HB-EGF in the regulation of angiogenesis in vivo was shown.Thus, the antigen binding proteins as described herein can be used fortreating diseases associated with or caused by angiogenesis, e.g.,cancerous or non-cancerous diseases.

For example HB-EGF antigen binding proteins as described herein mayinterfere with the communication between smooth muscle cells (SMCs) andendothelial cells, a fundamental process in the development andfunctionality of blood vessels in angiogenesis, e.g., tumorangiogenesis.

Furthermore, these antigen binding proteins may at least partiallyinhibit the HB-EGF induced expression and release of VEGF from SMCswhich acts subsequently as a powerful endothelial mitogen. Similarly,VEGF increases HB-EGF production in endothelial cells which thereforeconstitutes a pro-angiogenic feedback loop consisting of these twoimportant ligands that can be disrupted by administering the HB-EGFantigen binding proteins as described herein. Recently, and in additionto VEGF, further critical pro-angiogenic constituents such asangiopoetin 1 and 2 (Ang-1 & 2) and their receptor TIE-2 as well as thepotent smooth muscle cell GPCR stimulus angiotensin II (ATII) wereidentified. Interestingly, HB-EGF is a critical mediator of ATII-inducedEGFR transactivation and downstream upregulation of VEGF and Ang-2 inendothelial cells. In vivo, ATII induces angiogenesis in anHB-EGF-dependent manner and enhanced the angiogenic activity of VEGF.These findings support that in parallel to VEGF, HB-EGF is able toactivate additional angiogenic pathways via Ang2. Therefore, aberrantangiogenesis or cancer in a mammal may be treated with theadministration of an effective amount of the HB-EGF antigen bindingprotein described herein.

Besides its role as an important regulator of angiogenic processes incancer, various non-cancer indications with activated angiogenicpathways and the requirement of collateral blood flow are putativedisease areas for HB-EGF antigen binding protein therapy.

Chronic inflammatory diseases (e.g., nephritis, COPD, inflammatory boweldisease) including immune system mediated “inflammatory reactions”(e.g., graft versus host disease, transplant rejection, restenosis),metabolic diseases (e.g., diabetes), or chronic hypoxic conditions areoften characterized by hyper- and/or neo-vascularization (e.g., chroniculcers). The described HB-EGF antigen binding proteins may at leastpartially inhibit the essential process for angiogenesis—the recruitmentof vascular smooth muscle cells by endothelial cells induced by HB-EGFeither produced by inflammatory cells or upregulated by hypoxia—andrepresent therefore an attractive route for interventional therapy ofexcessive/pathological vascularization.

In obese subjects, increased levels of HB-EGF derived from theaccumulated fat which contribute to the higher incidence of vasculardisease in obesity, can be neutralized by the HB-EGF antigen bindingproteins described herein. A therapeutic intervention with an HB-EGFantigen binding protein may act therefore directly as ananti-adipocytokine in interrupting the adipovascular axis.

In fibroblasts, HB-EGF significantly downregulates elastin mRNA viaactivation of epidermal growth factor receptor. This effect provides anavenue of intervention in the development of lung fibrosis.

Moreover HB-EGF is a mitogen of keratinocytes involved in thepathogenesis of inflammatory diseases. In addition, expression of HB-EGFand co-localization with the EGFR may play an important role in theearly pathogenesis of psoriasis which opens a potential therapeuticwindow in the treatment of cutaneous inflammatory diseases andspecifically in the early treatment of psoriasis with the claimedantigen binding proteins.

Targeting HB-EGF with the described antigen binding proteins may alsoresult in a treatment option for patients with proliferativevitreoretinopathy (PVR), since the development of PVR is accompanied bya significant upregulation of HB-EGF in PVR retinas. In addition HB-EGFexpression in fibroproliferative tissue and its stimulatory effect onglial cell proliferation, chemotaxis, and VEGF secretion suggest thatHB-EGF may be a factor mediating glial cell responses during PVR. Theseprinciples can also be applied to further angiogenesis-dependent eyediseases such as age-related and non age-related macular degeneration,diabetic retinopathy, rubeotic glaucoma, interstitial keratitis,retinopathy of prematurity and corneal graft failure.

GPCRs such as adrenoceptors and angiotensin receptors have been linkedto the pathogenesis of hypertension due to their vasoconstrictive andgrowth promoting abilities. Since HB-EGF is a critical mediator of thesepathways, neutralizing antigen binding proteins are suitable fortargeted intervention in hypertensive disorders such as cardiachypertrophy and congestive heart failure, kidney failure or stroke.

HB-EGF, a potent mitogen and chemoattractant for smooth muscle cells(SMCs), was detected in atherosclerotic plaques of coronary arterieswith intimal thickening and produced by SMCs and macrophages. Remnantlipoproteins which are known causative agents of atherosclerosis werefurthermore shown to induce SMC proliferation via HB-EGF mediated EGFRtransactivation. Therefore, the HB-EGF antigen binding proteins asdescribed herein represent selective and efficient agents targetingatherosclerosis by blocking critical smooth muscle cell functions. Inaddition restenosis after percutaneous coronary intervention which ischaracterized by proliferation of smooth muscle cells might also beprevented by specific neutralization of HB-EGF function.

Thus, the HB-EGF antigen binding proteins as described herein can beused to treat a variety of diseases, including non-malignantproliferative diseases such as aberrant angiogenesis, leiomyoma (uterinefibrosis), benign smooth muscle cell tumors, glomerulosclerosis(hyperproliferation of mesangial cells), smooth muscle cell hyperplasia,atherosclerosis (hyperproliferation of vascular smooth muscle cells),rubeosis; neovascular glaucoma, diabetic retinopathy, diabeticblindness, macular degeneration, rheumatoid arthritis, cardiachypertrophy, psoriasis, and the like.

In a further aspect, these HB-EGF antigen binding proteins can be usedto treat disorders associated with or accompanied by a disturbed, e.g.,pathologically enhanced growth factor receptor activation.

In another aspect, this enhanced growth factor receptor activation maybe associated with or caused by a pathological increase in the activityof a G protein and/or a G protein coupled receptor. It should be notedthat disorders that are associated with or accompanied by a disturbed,e.g., pathologically enhanced growth factor receptor activation andwhich are may be associated with or caused by a pathological increase inthe activity of a G protein and/or a G protein coupled receptor, can bedelimited from other disorders characterized by an enhanced activity ofgrowth factor receptor activation in that a transactivation of thegrowth factor receptor via G protein coupled receptor takes place.

2. Diagnostic Methods

The HB-EGF antigen binding molecules as described herein may be used ina method for detecting cancer cells in a test sample that includescontacting the test sample with the antigen binding molecule anddetecting whether the antibody binds to a cell expressing proHB-EGF orHB-EGF molecules in the sample. The degree to which binding occurs canbe assessed by use of a control sample. The test and control samplescan, for example, be blood, serum, ascites, pleural effusion,cerebro-spinal fluid, tissue, cell, urine, lymph, saliva, milk or othersamples. Such a control sample can be a non-cancerous sample of thefluid or tissue or cell type. In some embodiments, the control sample isa non-cancerous sample obtained from the same subject as the testsample. “Subject” or “patient” refers to a mammal, preferably a human,in need of treatment for or detection of a condition, disorder ordisease.

The binding of antibody to components of the test sample can be detectedby detecting a reporting molecule, imaging agent or label that is boundor can be selectively bound to a antigen binding protein as describedherein. These HB-EGF antigen binding proteins can have one or morereporter molecules, labels or imaging agents. A reporter molecule isdefined as any moiety that may be detected using an assay. Non-limitingexamples of reporter molecules that have been conjugated to antibodiesinclude enzymes, radiolabels, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,luminescent molecules, photoaffinity molecules, colored particles orligands, such as biotin. The reporting molecule can provide a detectablesignal. For example, the detectable signal can be a fluorescent,phosphorescent, chemiluminescent, electrochemiluminescent,electrochemical, color change or enzymatic signal. Anti-HB-EGFantibodies provided herein can be used as capturing antibodies forELISA-based detection of the growth factor or immunohistochemicalanalysis of tissue samples as shown in FIG. 39.

In some embodiments, the reporting molecule is covalently bound to aantigen binding protein as described herein. In other embodiments, thereporting molecule can be selectively bound to the HB-EGF antigenbinding protein as described herein. Such selective binding of areporting molecule to the described antigen binding protein can beaccomplished by a secondary antibody with the label covalently boundthereto, where the secondary antibody selectively binds to an HB-EGBantigen binding protein as described herein.

3. Methods of Treatment: Pharmaceutical Formulations, Routes ofAdministration

Treatment of, or treating, cancer is intended to include the alleviationof or diminishment of at least one symptom typically associated with thedisease. The treatment also includes alleviation or diminishment of morethan one of the associated symptoms. The treatment may cure the cancer,e.g., it may substantially eliminate the cancer cells and/or arrest thegrowth of the cancerous tumor. Alternatively, treatment may slow theprogression of the cancer.

Anti-cancer activity can be evaluated against varieties of cancers orcancer cells using methods available to one of skill in the art.Anti-cancer activity, for example, can be determined by identifying theLD₁₀₀ or ED₅₀ of a preparation of the antigen binding proteins describedherein that prevents the growth of a cancer. In one embodiment,anti-cancer activity is the amount of antibody that kills 50% or 100% ofthe cancer cells when measured using standard dose response methods.

The HB-EGF antigen binding proteins as described herein can beadministered alone, or in combination with antibodies, chemotherapeuticdrugs or radiation therapy. Pharmaceutical compositions according to theinvention may be administered as monotherapy or in combination withanother pharmaceutical composition, preferably comprising anotheranti-neoplastic agent, in particular Cisplantin or Avastin. For example,the described HB-EGF antigen binding proteins can be co-administeredwith anti-tumor antibodies, (e.g., chimeric, humanized or humananti-tumor antibodies), with antibodies that specifically bind to VEGFto further inhibit tumor angiogenesis, or with antibodies thatspecifically bind to a receptor tyrosine kinase such as HER2, HER4 orEGFR and therefore further inhibit tumor cell proliferation. In FIG. 38,the synergistic effect of an anti-EGFR antibody and antiHB-EGFantibodies could be shown for both therapeutic antiHB-EGF antibodiestested. Further, the HB-EGF antigen binding proteins described hereinmay be co-administered with other anti-tumor agents. Specific examplesof anti-tumor agents which can be co-administered with the antibodiesprovided herein include, for example, gefitinib, lapatinib, sunitinib,pemetrexed, bevacisumab, cetuximab, imatinib, trastuzumab, alemtuzumab,rituximab, erlotinib, bortezomib and the like. Any other anti-canceragent or drug that can inhibit cancer or tumor cell proliferation canalso be used in the compositions as described and claimed herein. Forexample, the claimed compositions can include chemotherapeutic agentssuch as capecitabine, daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide, trimetrexate, teniposide,cisplatin, avastin and diethylstilbestrol (DES). See, generally, TheMerck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228,Berkow et al., eds., Rahway, N.J. When used with the described HB-EGFantigen binding proteins, such chemotherapeutic agents may be usedindividually (e.g., 5-FU and an antibody), sequentially (e.g., 5-FU andan antibody for a period of time followed by MTX and an antibody), or incombination with one or more other such chemotherapeutic agents (e.g.,5-FU, MTX and an antibody, or 5-FU, radiotherapy and an antibody).

Also provided is a method of evaluating a therapeutically effectivedosage for treating a cancer with an antibody having an amino acidsequence disclosed herein, that includes determining the LD₁₀₀ or ED₅₀of the HB-EGF antigen binding protein preparation in vivo or in vitro.Such a method permits calculation of the approximate amount of antigenbinding protein needed per volume to inhibit cancer cell growth ormetastasis by about 10% to 100%, or about 20% to 100%, or about 25% to100%, or about 30% to 100%, or about 40% to 100%, or about 50% to 100%.In some embodiments, less than 100% inhibition of cancer cell growth ormetastasis is observed. For example, cancer cell growth and/ormetastasis is inhibited by about 90%, 80%, 70%, 60%, or 50%. Percentageinhibition can be determined, for example, by administration of theantigen binding protein preparation to SCID or nu/nu mice available inthe art wherein tumor cells have been introduced and/or by standardmethods using cultured cancer cells (see, FIG. 38B). Several methods aredescribed in the Examples.

Also included are sterile pharmaceutical formulations of the HB-EGFantigen binding proteins as described herein that are useful astreatments for diseases including, for example, cancer and/or aberrantangiogenesis. Such formulations would inhibit the binding of HB-EGF toits receptor, e.g., EGFR or to HER4, thereby effectively treatingpathological conditions where, for example, serum, cellular or tissueHB-EGF is abnormally elevated or where its receptors, e.g., EGFR orHER4, are abnormally active. As illustrated herein the HB-EGF antigenbinding proteins possess adequate affinity to potently neutralize HB-EGFand to modulate the signaling events associated with the HB-EGFreceptors.

The HB-EGF antigen binding proteins as described herein are preferablyhumanized antigen binding proteins. Administration of these humanizedantigen binding proteins reduce the probability of a negative sideeffect. Moreover, these antigen binding proteins are stable in vivo, forexample, because they are recognized as normal human products, therebyminimizing the risk of immune system responses. Moreover, these antigenbinding proteins are not prone to proteolytic destruction, improvingtheir circulating half-lives. Hence, the HB-EGF preparations asdescribed herein have an excellent half-life in vivo so thatadministration in humans is comparatively infrequent. Such a prolongedduration of action may allow for less frequent and more convenientdosing schedules by alternate parenteral routes such as subcutaneous orintramuscular injection.

Sterile formulations can be created, for example, by filtration throughsterile filtration membranes, prior to or following lyophilization andreconstitution of the antibody. The antigen binding proteins asdescribed herein are ordinarily stored in lyophilized form or insolution. Therapeutic antibody compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having an adapter that allows retrieval of theformulation, such as a stopper pierceable by a hypodermic injectionneedle.

The route of administration of the HB-EGF antigen binding proteins arein accord with known methods, e.g., injection or infusion byintravenous, subcutaneous, intradermal, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial, intrathecal, intravesical,intra-cavernous, inhalation, intralesional routes, or by sustainedrelease systems as noted below. In some embodiments, the antigen bindingproteins as described herein are administered continuously by infusionor by bolus injection.

The HB-EGF antigen binding proteins, as described herein, can beprepared in a mixture with a pharmaceutically acceptable carrier. Thistherapeutic composition can be administered intravenously or through thenose or lung, preferably as a liquid or powder aerosol (lyophilized).The composition may also be administered parenterally or subcutaneouslyas desired. When administered systemically, the therapeutic compositionshould be sterile, pyrogen-free and in a parenterally acceptablesolution with consideration for what are physiologically acceptable pHvalues, isotonicity, and stability. These conditions are known to thoseskilled in the art. Briefly, dosage formulations of the compoundsdescribed herein are prepared for storage or administration by mixingthe compound having the desired degree of purity with physiologicallyacceptable carriers, excipients, or stabilizers. Such materials arenon-toxic to the recipients at the dosages and concentrations employed,and include buffers such as TRIS HCl, phosphate, citrate, acetate andother organic acid salts; antioxidants such as ascorbic acid; lowmolecular weight (less than about ten residues) peptides such aspolyarginine, proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone;amino acids such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;counterions such as sodium and/or nonionic surfactants such as TWEEN,PLURONICS or polyethyleneglycol.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice as described in Remington: TheScience and Practice of Pharmacy (20^(th) ed, Lippincott Williams &Wilkens Publishers (2003)). For example, dissolution or suspension ofthe active compound in a vehicle such as water or naturally occurringvegetable oil like sesame, peanut, or cottonseed oil or a syntheticfatty vehicle like ethyl oleate or the like may be desired. Buffers,preservatives, antioxidants and the like can be incorporated accordingto accepted pharmaceutical practice.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles, films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., 1981, J. Biomed Mater. Res. 15:167-277 andLanger, 1982, Chem. Tech. 12:98-105, or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,Biopolymers 22:547-556), non-degradable ethylene-vinyl acetate (Langeret al., supra), degradable lactic acid-glycolic acid copolymers such asthe LUPRON Depot™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation through disulfideinterchange, stabilization may be achieved by modifying sulfhydrylresidues, lyophilizing from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

Sustained-released compositions also include preparations of crystals ofthe antigen binding protein suspended in suitable formulations capableof maintaining crystals in suspension. These preparations when injectedsubcutaneously or intraperitoneally can produce a sustained releaseeffect. Other compositions also include liposomally entrappedantibodies. Liposomes containing such antibodies are prepared by methodsknown per se: U.S. Pat. No. DE 3,218,121; Epstein et al., 1985, Proc.Natl. Acad. Sci. USA 82:3688-3692; Hwang et al., 1980, Proc. Natl. Acad.Sci. USA 77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949;142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324.

The dosage of the antigen binding protein formulation for a givenpatient will be determined by the attending physician taking intoconsideration various factors known to modify the action of drugsincluding severity and type of disease, body weight, sex, diet, time androute of administration, other medications and other relevant clinicalfactors. Therapeutically effective dosages may be determined by eitherin vitro or in vivo methods.

An effective amount of the antigen binding proteins, described herein,to be employed therapeutically will depend, for example, upon thetherapeutic objectives, the route of administration, and the conditionof the patient. Accordingly, it is preferred for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 0.001 mg/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Typically, the clinician will administer thetherapeutic antigen binding protein until a dosage is reached thatachieves the desired effect. The progress of this therapy is easilymonitored by conventional assays or as described herein.

It will be appreciated that administration of therapeutic entities inaccordance with the compositions and methods herein will be administeredwith suitable carriers, excipients, and other agents that areincorporated into formulations to provide improved transfer, delivery,tolerance, and the like. These formulations include, for example,powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as Lipofectin™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Any of the foregoing mixtures may be appropriate in treatments andtherapies involving the HB-EGF antigen binding protein as describedherein, provided that the active ingredient in the formulation is notinactivated by the formulation and the formulation is physiologicallycompatible and tolerable with the route of administration. See, alsoBaldrick P. “Pharmaceutical excipient development: the need forpreclinical guidance,” 2000, Regul. Toxicol. Pharmacol. 32:210-218;Wang, “Lyophilization and development of solid protein pharmaceuticals,”2000, Int. J. Pharm. 203:1-60; Charman W N “Lipids, lipophilic drugs,and oral drug delivery-some emerging concepts,” J. Pharm. Sci0.89:967-978; Powell et al., 1998, “Compendium of excipients forparenteral formulations,” PDA J. Pharm. Sci. Technol. 52:238-311 and thecitations therein for additional information related to formulations,excipients and carriers well known to pharmaceutical chemists.

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the teachings herein.

EXAMPLES A. Example 1 Generation of Immunogen

HB-EGF including EGF-like domain (aa 1-149) was amplified from apcDNA3-VSV-HB-EGF expression construct (Prenzel et al., 1999, supra) andcloned into an expression vector that provides an in-frame 6H is tag atthe carboxyl-terminus (pcDNA 3.1 myc-his, InVitrogen). This HB-EGFimmunogen with C-terminal myc(HIS)6 tag was expressed in HEK293 cellsand purified by a two step purification on Ni-NTA sepharose (AmershamPharmacia) and heparin sepharose (Sigma).

The HB-EGF portion of the immunogen had the following sequence (SEQ IDNO:1077).

  1 MKLLPSVVLK LFLAAVLSAL VTGESLERLR RGLAAGTSNP  41DPPTVSTDQL LPLGGGRDRK VRDLQEADLD LLRVTLSSKP  81QALATPNKEE HGKRKKKGKG LGKKRDPCLR KYKDFCIHGE 121CKYVKELRAP SCICHPGYHG ERCHGLSLP

B. Example 2 Immunization of Xenomouse Mice and Titers Observed

Monoclonal antibodies against HB-EGF were developed by sequentiallyimmunizing XenoMouse® mice (XenoMouse strains: XMG2 (humanIgG2-producing) and XM3C-1 (human IgG4-producing), Abgenix, Inc.Fremont, Calif.).

1. Immunization

XenoMouse animals were immunized via the footpad for all injections. Thetotal volume of each injection was 50 μl per mouse, 25 μl per footpad.

For both cohort 1 (10 XMG2 mice) and Cohort 2 (10 XM3C-1), the initialimmunization was with 10 μg of HB-EGF protein admixed 1:1 (v/v) withTITERMAX GOLD® (Sigma, Oakville, ON) per mouse. The subsequent fourboosts were made with 10 μg of HB-EGF protein admixed 1:1 (v/v) with 100μg alum gel (Sigma, Oakville, ON) in pyrogen-free D-PBS. The fifth boostconsisted of 10 μg of HB-EGF protein admixed 1:1 (v/v) with TITERMAXGOLD®. The sixth and seventh injection consisted of 10 μg of HB-EGFprotein admixed 1:1 v/v with 100 μg alum gel. A final boost was madewith 10 μg of HB-EGF protein in pyrogen-free D-PBS, without adjuvant.The XenoMouse mice were immunized on days 0, 4, 8, 14, 18, 21, 25, and28 for this protocol and fusions were performed on day 32. The twobleeds were made through Retro-Orbital Bleed procedure on day 16 afterthe fourth boost, on day 23 after the sixth boost.

2. Selection of Animals for Harvest by Titer

Anti-HB-EGF antibody titers in the serum from immunized XenoMouse micewere determined by ELISA. Briefly, the HB-EGF protein (2 μg/ml) wascoated onto Costar Labcoat Universal Binding Polystyrene 96-well plates(Corning, Acton, Mass.) overnight at 4° C. in Antigen Coating Buffer(0.1 M Carbonate Buffer, pH 9.6 NaHCO₃ (MW 84) 8.4 g/L). The next day,the plates were washed one time with washing buffer (0.05% Tween 20 in1×PBS) using a Biotek plate washer. The plates were then blocked with200 μl/well blocking buffer (0.5% BSA, 0.1% Tween 20, 0.01% Thimerosalin 1×PBS) and incubated at room temperature for 1 hour. After theone-hour blocking, the plates were washed one time with washing bufferusing a Biotek plate washer. Sera from either the HB-EGF proteinimmunized XenoMouse mice or naïve XenoMouse animals were titrated in0.5% BSA/PBS buffer at 1:3 dilutions in duplicate from a 1:100 initialdilution. The last well was left blank. These plates were incubated atroom temperature for 2 hours, and the plates were then washed threetimes with washing buffer using a Biotek plate washer. A goat anti-humanIgG Fc-specific horseradish peroxidase (CALTAG, Cat NO, H10507)conjugated antibody was added at a final concentration of 1:2000 inblocking buffer and incubated for 1 hour at room temperature. The plateswere washed three times with washing buffer using a Biotek plate washer.After washing, the plates were developed with the addition of TMBchromogenic substrate (BioFx BSTP-0100-01) for 10-20 minutes or untilnegative control wells start to show color. Then the ELISA was stoppedby the addition of Stop Solution (650 nM Stop reagent for TMB (BioFxBSTP-0100-01), reconstituted with 100 ml H₂O per bottle). The specifictiter of each XenoMouse animal was determined from the optical densityat 650 nm and is shown in TABLE 1 below. The titer value is thereciprocal of the greatest dilution of sera with an OD reading two-foldthat of background. Therefore, the higher the number, the greater wasthe humoral immune response to HB-EGF.

TABLE 1 Group 1, fp, XMG2, 10 mice, Group 2, fp, XM3C-1, 10 mice, After4 inj. After 6 inj. After 4 inj. After 6 inj. Mouse ID Reactivity toHB-EGF Mouse ID Reactivity to HB-EGF P4721  3,800 59,000 P2664  35 2,300P4722  6,500 68,000 P2666  20 750 P4723  2,500 43,000 P2669  30 850P4724  2,400 22,000 P26610 75 1,900 P4725  2,400 71,000 P2892  60 550P4726  450 58,000 P2894  60 1,400 P4727  1,600 20,000 P2895  95 2,600P4728  2,700 61,000 P2896  45 2,500 P4729  800 61,000 P3672  50 2,000P47210 5,700 78,000 P3678  60 1,800 NC <100 <100 NC <100 <100 PC <100<100 PC 35 20

C. Example 3 Hybridoma Generation and Primary Screen for Binders

Immunized mice were sacrificed and the lymph nodes were harvested andpooled from each cohort. The lymphoid cells were dissociated by grindingin DMEM to release the cells from the tissues, and the cells weresuspended in DMEM. The cells were counted, and 0.9 ml DMEM per 100million lymphocytes was added to the cell pellet to resuspend the cellsgently but completely. Using 100 μl of CD90+ magnetic beads per 100million cells, the cells were labeled by incubating the cells with themagnetic beads at 4° C. for 15 minutes. The magnetically-labeled cellsuspension containing up to 10⁸ positive cells (or up to 2×10⁹ totalcells) was loaded onto a LS+ column and the column washed with DMEM. Thetotal effluent was collected as the CD90-negative fraction (most ofthese cells were expected to be B cells).

The fusion was performed by mixing washed enriched B cells from aboveand nonsecretory myeloma P3X63Ag8.653 cells purchased from ATCC, cat.#CRL 1580 (Kearney et al, 1979, J. Immunol. 123:1548-1550) at a ratio of1:1. The cell mixture was gently centrifuged at 800 g. After completeremoval of the supernatant, the cellular pellet was treated with 2-4 mlof Pronase solution (CalBiochem, cat. #53702; 0.5 mg/ml in PBS) for nomore than 2 minutes. Then, 3-5 ml of FBS was added to stop the enzymeactivity and the suspension was adjusted to 40 ml total volume usingelectro cell fusion solution, ECFS (0.3 M Sucrose, Sigma, Cat# S7903,0.1 mM Magnesium Acetate, Sigma, Cat# M2545, 0.1 mM Calcium Acetate,Sigma, Cat# C4705). The supernatant was removed after centrifugation andthe cells were resuspended in 40 ml ECFS. This wash step was repeatedand the cells again were resuspended in ECFS to a concentration of 2×10⁸cells/ml.

Electro-cell fusion was performed using a fusion generator, modelECM2001, Genetronic, Inc., San Diego, Calif. The fusion chamber sizeused was 2.0 ml, using the following instrument settings: Alignmentcondition: voltage: 50 V, time: 50 seconds; membrane breaking at:voltage: 3000 V, time: 30 μsec; post-fusion holding time: 3 seconds.

After electro-cell fusion, the cell suspensions were carefully removedfrom the fusion chamber under sterile conditions and transferred into asterile tube containing the same volume of Hybridoma Culture Medium(DMEM (JRH Biosciences), 15% FBS (Hyclone), supplemented withL-glutamine, pen/strep, OPI (oxaloacetate, pyruvate, bovine insulin)(all from Sigma) and IL-6 (Boehringer Mannheim)). The cells wereincubated for 15-30 minutes at 37° C., and then centrifuged at 400 g forfive minutes. The cells were gently resuspended in a small volume ofHybridoma Selection Medium (Hybridoma Culture Medium supplemented with0.5×HA (Sigma, cat. # A9666)), and the volume was adjusted appropriatelywith more Hybridoma Selection Medium, based on a final plating of 5×10⁸B cells total per 96-well plate and 200 μL per well. The cells weremixed gently and pipetted into 96-well plates and allowed to grow. Onday 7 or 10, one-half the medium was removed, and the cells were re-fedwith Hybridoma Selection Medium.

After 14 days of culture, hybridoma supernatants from the cohort #1 andcohort #2 were screened for HB-EGF-specific monoclonal antibodies byELISA. In the Primary screen, the ELISA plates (Fisher, Cat. No.12-565-136) were coated with 50 μL/well of HB-EGF protein (2 μg/ml) inCoating Buffer (0.1 M Carbonate Buffer, pH 9.6, NaHCO₃ 8.4 g/L), thenincubated at 4° C. overnight. After incubation, the plates were washedwith Washing Buffer (0.05% Tween 20 in PBS) one time. 200 μL/wellBlocking Buffer (0.5% BSA, 0.1% Tween 20, 0.01% Thimerosal in 1×PBS)were added and the plates were incubated at room temperature for 1 hour.After incubation, the plates were washed with Washing Buffer one time.Aliquots (50 μL/well) of hybridoma supernatants and positive andnegative controls were added, and the plates were incubated at roomtemperature for 2 h. The positive control used throughout was serum fromthe relevant HB-EGF protein-immunized XenoMouse mouse and the negativecontrol was serum from a KLH-immunized relevant strain of XenoMousemouse. After incubation, the plates were washed three times with WashingBuffer. 100 μL/well of detection antibody goat anti-huIgGFc-HRP (CaltagInc., Cat. No. H10507, using concentration was 1:2000 dilution) wasadded and the plates were incubated at room temperature for 1 hour.After incubation, the plates were washed three times with Washing Bufferand 100 μl/well of TMB (BioFX Lab. Cat. No. TMSK-0100-01) was added.Plates were then allowed to develop for about 10 minutes (until negativecontrol wells barely started to show color). 50 μl/well stop solution(TMB Stop Solution (BioFX Lab. Cat. No. STPR-0100-01) was then added andthe plates were read on an ELISA plate reader at a wavelength of 450 nm.

The old culture supernatants from the positive hybridoma cells growthwells based on primary screen were removed and the HB-EGF positivehybridoma cells were suspended with fresh hybridoma culture medium andwere transferred to 24-well plates. After 2 days in culture, thesesupernatants were ready for a secondary confirmation screen. In thesecondary confirmation screen, the positives in the first screening werescreened by ELISA (described as above) with two sets of detectiveantibodies: goat anti-huIgGFc-HRP (Caltag Inc., Cat. No. H10507, usingconcentration was 1:2000 dilution) for human gamma chain detection andgoat anti-hIg kappa-HRP (Southern Biotechnology, Cat. No. 2060-05) forhuman kappa light chain detection in order to demonstrate that theantibody preparation was HB-EGF-specific and fully human in itscomposition. The two sets of ELISA procedures were identical to thedescriptions above except the two different detection antibodies wereused separately.

In parallel with the secondary confirmation screen, the counter ELISAscreen was performed to exclude those antibodies that respond tomyc(his)6 tag. The ELISA procedures were identical to the descriptionsabove except the coated with irrelevant myc(his)6 tag protein(ML-myc(his)6 protein) instead of coating with HB-EGF myc(his)6 protein.

After the secondary confirmation and the counter ELISA screen, 49 fullyhuman IgG/kappa HB-EGF specific monoclonal antibodies were identifiedfrom cohorts 1 and 2.

D. Example 4 Scale-Up and Testing of Antibodies in Functional Assays

This Example describes the identification of hybridoma cell lines thatproduce anti-HB-EGF antibodies with affinity for HB-EGF.

1. FACS Detection of Hybridoma-Produced Anti-HB-EGF Antibodies Bound toHB-EGF Expressing Cell-Lines

HB-EGF expression on cell-lines was determined by FACS analysis. Toperform this analysis 2×10⁵ selected HB-EGF-expressing cells wereharvested with 10 mM EDTA in PBS, resuspended in FACS-buffer (PBS, 3%FCS, 0.4% azide) and seeded on a 96-well round bottom plate. Aftercentrifugation for 3 minutes at 1000 rpm to remove supernatant, thecells were resuspended in hybridoma-derived anti-HB-EGF antibodydilution (100 μl/well) and incubated at 4° C. for 45 min. The cells werewashed twice with FACS buffer and resuspended with secondary antibody(100 μl/well) donkey-anti-human-PE (Jackson) diluted 1:100 in FACSbuffer. The cell suspensions were incubated at 4° C. in the dark for 30min, washed twice with FACS buffer and analyzed (FACS, Beckman Coulter).

The results of these assays are provided in TABLES 2 and 3 below, whichprovide the fluorescence mean values for each FACS assay. As illustratedin TABLES 3 and 4, substantially no HB-EGF expression was detected inCHO control cells that do not express HB-EGF. However, when HB-EGF isrecombinantly overexpressed in CHO cells, several monoclonal antibodypreparations exhibit significant binding to those HB-EGF-expressingcells. Binding to MDA-MB231, SCC-9 and COS-7 cells was somewhat variablebut an antibody preparation that bound to one HB-EGF-expressing celltype typically bound to another. As shown in TABLE 2, several antibodypreparations, including, for example, the U2-24, U2-5, U2-19, U2-21,U2-15 and U2-42 antibody preparations exhibited strong binding toHB-EGF-expressing CHO cells. Similarly, antibody preparations U2-39,U2-26, U2-44, U2-45 and U2-48 also exhibited good binding toHB-EGF-expressing CHO cells.

TABLE 2 FACS Analysis Of Anti-HB-EGF Antibody Supernatants (Cohort 1)CHO control cells vs. HB- Cell Lines Endogenously EGF-expressing CHOcells Expressing HB-EGF Antibody CHO-K1 CHO-HB-EGF MDA-MB231 SCC-9 COS-7KLH 0.2 0.3 0.4 0.2 0.4 U2-18 0.3 154 3.1 1.2 1.6 U2-68 0.2 238 1.7 0.61.1 U2-24 0.2 360 2 0.6 6.7 U2-14 0.3 132 2.8 0.8 1.1 U2-1 0.2 64 1.80.7 3.1 U2-32 0.2 76 1.7 1.2 1.8 U2-40 0.2 137 2.1 1.3 1.1 U2-5 0.3 3716.9 1.7 2.2 U2-8 0.3 148 3.7 1.4 1.4 U2-13 0.2 136 0.4(2.6) 0.5 0.8U2-17 0.2 170 1.6 0.6 1.4 U2-19 0.4 347 5.5 1.8 6.1 U2-38 0.3 30 3.9 0.91.4 U2-21 0.2 344 3.9 3.4 2.1 U2-15 0.2 370 3 0.6 0.9 U2-16 0.2 197 1.50.4 0.9 U2-30 0.3 273 3.5 1 3.4 U2-44 0.3 5.6 0.8 0.2 5.2 U2-42 1.2 2773.3 0.5 6.7 U2-36 0.9 112 1.6 0.3 4.9 U2-22 0.3 221 1.1 0.2 4.9 U2-560.2 38 0.5 0.2 1.8 Neg 0.2 0.2 0.3 Pos (goat 0.2 80 3 aHB)

TABLE 3 FACS Analysis Of Anti-HB-EGF Antibody Supernatants (Cohort 2)CHO control cells vs. HB- Cell Lines Endogenously EGF-expressing CHOcells Expressing HB-EGF Antibody CHO-K1 CHO-HB-EGF MDA-MB231 SCC-9 COS-7U2-63 0.2 36(0.4) 0.5 0.2 0.6 U2-54 0.3 49 0.6 0.2 2.4 U2-65 0.2 1.9 0.40.2 0.3 U2-10 0.2 2.3 0.5 0.2 1.2 U2-53 0.2 144 0.5 0.2 0.6 U2-66 0.2149 0.5 0.2 0.5 U2-61 0.4 28 0.4 0.2 1.4 U2-67 0.2 82 0.5 0.2 0.6 U2-280.3 198 1 0.2 3.2 U2-2 0.2 127 0.5 0.2 0.5 U2-62 0.2 47 0.5 0.2 0.6U2-39 0.2 205 2.6 0.6 8.3 U2-3 0.2 168 0.9 0.2 2.5 U2-43 0.4 185 0.8 0.21.7 U2-34 0.7 125 1.2 0.3 6.4 U2-26 0.2 242 1.5 0.3 4.7 U2-41 0.3 1861.2 0.2 3.3 U2-44 0.4 248 1.2 0.2 2.2 U2-45 0.6 248 1.4 0.2 6.4 U2-570.2 201 0.7 0.2 0.5 U2-12 0.6 1.2 1.2 0.5 1.6 U2-46 0.4 129 1 0.2 2.5U2-48 0.4 273 1.3 0.3 2.1 U2-6 0.5 108 3.9 1.5 5.9 U2-58 0.2 277 0.9 0.20.4 U2-51 0.5 176 1.1 0.2 3.6 U2-60 0.2 3.6 0.4 0.2 0.3 Neg 0.2 0.2 0.3Pos (goat 0.2 80 3 aHB)

2. Inhibition of HB-EGF-Induced EGFR Tyrosine Phosphorylation

The following protocol was used to identify which anti-HB-EGF antibodypreparations inhibit HB-EGF-induced epidermal growth factor receptortyrosine phosphorylation. Such experiments further characterize theantibodies and help identify which hybridoma cell lines produce antibodythat inhibit HB-EGF activity and then should be cloned and expanded.

40000 SCC9 human squamous cancer cells were seeded on a 96-well plate in100 μl medium. Cells were starved in 60 μl serum free medium for 24 hr.A black Maxisorp 96-well plate was coated with 100 μl anti-EGFR antibody(2 μg/ml) overnight at 4° C. The coating solution was replaced by 300 μlblocking solution (PBS+0.5% BSA) without washing and left to incubate 2hours at room temperature. 10 μg/ml of IgG2-control (Sigma) oranti-HB-EGF hybridoma-derived antibodies were added to 20 ng/ml HB-EGF(R&D Systems) and preincubated for 30 minutes at 37° C. (volume: 40 μl).Cells were treated with medium alone or with the antibody/ligandsolution for 3 minutes at 37° C. The medium was removed and cells werelysed on ice with 100 μl Triton-X-100-based lysis buffer containing 1 mMPMSF, 10 μg/ml Aprotinin, 10 mM NaF and 2 mM Na-Orthovanadate. Theblocked Maxisorp plate was washed 6 times with PBS+0.05% Tween-20. 80 μlof the cell lysate was transferred directly to the washed Maxisorp plateand incubated overnight at 4° C. with gentle agitation. The plate waswashed 6 times with PBS+0.05% Tween-20, then 100 μl 4G10-biotin (UBI)diluted 1:4000 in dilution buffer (PBS+0.5% BSA+0.05% Tween-20+5 mMEDTA) was added to each well and incubated for 2 hours at roomtemperature. The plate was washed 6 times with PBS+0.05% Tween20 and 100μl AP-conjugated streptavidin (UBI) diluted 1:20000 in dilution buffer(PBS+0.5% BSA+0.05% Tween20+5 mM EDTA) was added to each well for 30minutes at room temperature. The plate was washed 6 times with PBS+0.05%Tween-20 and 100 μl AttoPhos substrate was added to each well. The platewas incubated for up to 3 hours at room temperature in the dark and thedeveloping fluorescence was monitored at 30, 90 and 180 min (Excitation:430 nm, emission: 580 nm).

The percent inhibition of HB-EGF-induced EGFR tyrosine phosphorylationwas calculated by reduction in the amount of phosphorylation observedwith IgG2-control (Sigma) by each hybridoma-derived anti-HB-EGF antibodypreparation.

Results for the different anti-HB-EGF antibody preparations are providedin FIGS. 23A and 23B. As illustrated, monoclonal antibody preparationsU2-18, U2-24, U2-19, U2-42, U2-39, U2-34, U2-45 and U2-6 stronglyinhibit EGFR tyrosine phosphorylation.

3. Anti-HB-EGF Antibody Preparations Inhibit LPA-Induced EGFRPhosphorylation

EGFR-dependent signaling pathways can be activated upon stimulation ofG-protein-coupled receptors (GPCR). Ligand activation of heterotrimericG proteins by interaction with a GPCR results in an intracellular signalthat induces the extracellular activity of a transmembranemetalloproteinase. Ligands that can activate the GPCR pathway includeLPA (lysophosphatidic acid), thrombin, carbachol, bombesin, andendothelin. Such activation leads to extracellular processing of atransmembrane growth factor precursor and release of the mature factorwhich, directly or through the proteoglycan matrix, interacts with theectodomain of EGFR and activates it through phosphorylation. See,Prenzel et al., 1999, Nature 402:884-888.

The anti-HB-EGF antibody preparations provided herein were tested toascertain whether they could inhibit EGFR tyrosine phosphorylationinduced by the GPCR ligand LPA in COS-7 cells, using the followingprocedure.

250.000 COS-7 cells were seeded on a 6-well plate, in 2 ml medium andcultured over night. Cells were starved in 1 ml serum free medium for 24hours. Following preincubation with antibodies (37° C., 1 h), cells werestimulated with 10 μM LPA (37° C., 3 min) and lysed on ice with 400 μlTriton-X-100-based lysis buffer containing 1 mM PMSF, 10 μg/mlAprotinin, 10 mM NaF and 2 mM Na-Orthovanadate. Followingimmunoprecipitation of the EGFR (340 μl lysate, 340 μl HNTG, 30 μl Prot.A Sepharose, 1.5 μl αEGFR (108.1, Prenzel et al., 1999, supra)) for 4hours in the coldroom precipitates were washed 3 times with 500 μl HNTGbuffer and run on a 7.5% SDS PAGE. Following transfer on anitrocellulose membrane (Schleicher & Schuell) the blot was probed withan antibody recognizing phosphotyrosine residues (primary ab 4G10(1:2000, Upstate biotechnology); secondary anti-mouse Ab 1:10000,Jackson laboratories). Reblot with sheep anti EGFR (Upstate technology)after stripping showed that equal amounts of the receptor wereprecipitated in each lane.

FIG. 24 provides a western blot illustrating which anti-HB-EGF antibodypreparations inhibit LPA-induced EGFR tyrosine phosphorylation. As shownin FIG. 24, the U2-19 and U2-42 anti-HB-EGF antibody preparationsstrongly inhibited LPA-induced EGFR phosphorylation. The U2-24anti-HB-EGF antibody preparation inhibited LPA-induced EGFRphosphorylation to a somewhat lesser extent.

E. Example 5 Hybridoma Cloning to Generate Monoclonal Antibodies

Based on the test results observed from experiments described in thepreceding Examples, of the 49 original isolates, 43 hybridoma cell lineswere selected for cloning by limiting dilution and furthercharacterization of their monoclonal antibody. The lines selected forcloning bound to HB-EGF-expressing cell lines as exhibited by FACSanalysis and inhibited HB-EGF-stimulation of EGF receptor tyrosinephosphorylation. For some hybridomas, insufficient antibody wasgenerated to run all the primary screening assays. This subset ofhybridomas was advanced to hybridoma cloning as well.

F. Example 6 Further Characterization of Antibody-Related Inhibition ofGPCR Induced Tyrosine Phosphorylation of EGFR

This Example provides further data showing that several anti-HB-EGFantibody preparations provided herein exhibit dose-dependent inhibitionof GPCR-induced EGF receptor tyrosine phosphorylation.

Inhibition by candidate antibody preparations U2-42, U2-39 and U2-45 wasexamined using different concentrations of these antibody preparationsand the following procedure.

150,000 cells (MDA-MB231, PPC1) were seeded on a 12-well plate in 1 mlmedium. Cells were starved in 500 μl serum free medium for 24 hr. Ablack Maxisorp 96-well plate was coated with 100 μl anti-EGFR antibody(2 μg/ml) overnight at 4° C. The coating solution was replaced by 300 μlblocking solution (PBS+0.5% BSA) without washing and left to incubate 2hours at room temperature. Cells were pre-incubated with 10 μg/mlanti-HB-EGF Abs for 30 minutes at 37° C. and then treated with the GPCRligands LPA (10 μM, PPC1 cells) or Thrombin (1 U/ml, MDA-MB231 cells)for 3 minutes at 37° C. The medium was removed and cells were lysed onice with 200 μl Triton-X-100-based lysis buffer containing 1 mM PMSF, 10μg/ml Aprotinin, 10 mM NaF and 2 mM Na-Orthovanadate. The blockedMaxisorp plate was washed 6× with PBS+0.05% Tween-20. Cell lysate wastransferred directly to a washed Maxisorp plate and incubated overnightat 4° C. with gentle agitation.

The plate was washed 6 times with PBS+0.05% Tween-20, then 100 μl4G10-biotin (UBI) diluted 1:4000 in dilution buffer (PBS+0.5% BSA+0.05%Tween-20+5 mM EDTA) was added to each well and incubated for 2 hours atroom temperature. The plate was washed 6 times with PBS+0.05% Tween-20and 100 μl AP-conjugated streptavidin (UBI) diluted 1:20000 in dilutionbuffer (PBS+0.5% BSA+0.05% Tween-20+5 mM EDTA) was added to each wellfor 30 minutes at room temperature. The plate was washed 6 times withPBS+0.05% Tween-20 and 100 μl Attophos substrate was added to each well.The plate was incubated for 3 hours at room temperature in the dark andthe developing fluorescence was monitored at 30, 90 and 180 min(Excitation: 430 nm, emission: 580 nm).

As shown in FIG. 26, inhibition of LPA-induced EGFR tyrosinephosphorylation was dose dependent—greater inhibition of EGFR tyrosinephosphorylation was observed as the amount of anti-HB-EGF antibody wasincreased.

FIG. 25 illustrates that candidate antibody preparations U2-42, U2-39and U2-45 also effectively inhibit thrombin-induced EGFR phosphorylationin MDA-MB231 cells. A dosage dependent inhibition is observed, withincreased inhibition as more anti-HB-EGF antibody is added. FIG. 26illustrates that inhibition of LPA-induced EGFR tyrosine phosphorylationin PPC-1 cells by anti-HB-EGF antibody preparations provided herein isdose dependent. As shown, greater inhibition of EGFR tyrosinephosphorylation was detected as the amount of anti-HB-EGF antibody wasincreased.

G. Example 7 Inhibition of GPCR-Induced MDA-MB231 Cell Migration byHuman Anti-HB-EGF Antibodies

Transmigration experiments were performed in order to investigatewhether the antibodies provided herein block cell migration induced bythe GPCR ligand Sphingosine-1-phosphate.

Serum-starved human breast cancer MDA-MB231 cells were preincubated withthe indicated antibody to the cell suspension for 45 min at 37° C.Thereafter, 500 ml cell suspension (50,000 cells) was placed in the topchamber of collagen I-coated transwells (BD Falcon, 8 μm pores). 750 mlmedium (MEM, amino acids, Na-Pyruvate, Pen.-Strept., 0.1% BSA) alone orcontaining the GPCR ligand Sphingosine-1-phosphate (R&D Systems) wasused in the bottom chamber. After migration for 8 hours at 37° C. cellswere fixed, stained with DAPI and cell nuclei were counted forstatistical evaluation.

The results for these MDA-MB231 cell migration assays using candidateanti-HB-EGF antibody preparations U2-42, U2-39 and U2-45 are provided inFIG. 27. As shown, anti-HB-EGF antibody preparation U2-42 inhibitedMDA-MB231 cell migration by about 70%; anti-HB-EGF antibody preparationU2-45 inhibited MDA-MB231 cell migration by about 100%; and anti-HB-EGFantibody preparation U2-39 inhibited MDA-MB231 cell migration by about100%. Hence, the ability of these anti-HB-EGF antibodies to inhibitMDA-MB231 cell migration is substantial.

H. Example 8 Inhibition of HB-EGF-Induced Migration of MCF-7 Cells byHuman Anti-HB-EGF Antibodies

Transmigration experiments were performed in order to investigatewhether the antibodies provided herein block cell migration that wouldotherwise be directly induced by HB-EGF. The results of these testshighlight which antibody preparations may be use for development asanti-metastatic cancer agents.

A 500 ml cell suspension of serum-starved human breast cancer MCF7 cells(50,000 cells) was placed in the top chamber of collagen I-coatedtranswells (BD Falcon, 8 μm pores). Aliquots of 750 ml medium (MEM,amino acids, Na-pyruvate, Pen.-Strept., 0.1% BSA) alone or containing 20ng/ml HB-EGF (R&D Systems) in the presence or absence of 10 μg/ml HB-EGFantibodies were placed in the bottom chamber. After incubation andmigration for 8 hours at 37° C., cells were fixed, stained with DAPI andcell nuclei were counted for statistical evaluation.

The results for these migration assays using candidate anti-HB-EGFantibody preparations U2-42, U2-39 and U2-45 are provided in FIG. 28. Asshown, anti-HB-EGF antibody preparation U2-42 inhibited MCF-7 cellmigration by about 55%; anti-HB-EGF antibody preparation U2-45 inhibitedMCF-7 cell migration by about 93%; and anti-HB-EGF antibody preparationU2-39 inhibited MCF-7 cell migration by about 98%. Hence, the ability ofthese anti-HB-EGF antibodies to inhibit MCF-7 cell migration issubstantial.

I. Example 9 Characteristics of Top 10 Anti-HB-EGF Antibodies

A summary of results is provided in TABLE 4, infra, for top 10hybridoma-derived antibody preparations in GPCR-induced triplemembrane-passing signal (TMPS) experiments. The TMPS experimentsinvolved LPA-stimulation in SCC9 cells, thrombin stimulation inMDA-MB231 cells, LPA stimulation of SkOV-8 cells andSphingosine-1-phosphate migration of MDA-MB231 cells. The data providedrepresent the percent inhibition of the TMPS transactivation signal thatwas observed when antibody preparations were used in the assays comparedto the same assay when no antibody was present. The top three antibodiesof each experiment are highlighted in bold letters.

TABLE 4 Percent Inhibition of TMPS Transactivation Signal TMPS MigrationAb SCC9.3 MDA-MB231 MDA-MB231 SkoV-8 SkoV-8 MDA-MB231 U2-39 56.9 126.6131.1 130.7 65.5 115 U2-45 58.5 136.8 95.7 99.4 75.1 104 U2-42 62.8123.2 125.6 110.7 73.1 69.9 U2-34 50.6 109.5 93.6 95.1 73   99.6 U2-4636.6 88.1 77.5 80.5 76.1 n.d. U2-19 n.d. n.d. n.d. n.d. n.d. 85 U2-26n.d. 143 91.8 106.1 31.4 55.9 U2-51 n.d. n.d. n.d. n.d. n.d. 85.3 U2-15n.d. 121.7 92.6 87 17.6 45.5 U2-22 n.d. 74.2 94.3 64.4 18.2 n.d.

J. Example 10 Inhibition of HB-EGF-Induced HER4 Tyrosine Phosphorylationby Human Anti-HB-EGF Antibodies

This Example shows that anti-HB-EGF antibody preparations inhibit HB-EGFinduced tyrosine phosphorylation of HER4. The following procedures wereemployed to assess the effects of anti-HB-EGF antibodies on HER4tyrosine phosphorylation.

125,000 cells T47D human breast cancer cells were seeded on a 24-wellplate in 500 μl medium. Cells were starved in 200 μl serum free mediumfor 24 hr. An R&D Systems Human Phospho-ErbB4 ELISA-Kit was used fordetection of HER4 tyrosine phosphorylation. A clear Maxisorp 96-wellplate was coated with 100 μl mouse anti-human-ErbB4 antibody (CaptureAntibody 1 μg/ml) overnight at room temperature. The coated Maxisorpplate was washed 6 times with PBS+0.05% Tween-20, the washing solutionwas replaced by 300 μl blocking solution (PBS+0.5% BSA) and incubated 2hours at room temperature. 50 μl serum-free medium with 5×-concentrationof anti-HB-EGF Abs (10 μg/ml) was incubated with 5×-concentration HB-EGF(20 ng/ml) for 30 min at 37° C., then added to each well (in duplicate).The medium was removed and cells were lysed on ice with 200 μlTriton-X-100-based lysis buffer containing 1 mM PMSF, 10 μg/mlAprotinin, 10 mM NaF and 2 mM Na-Orthovanadate. The blocked Maxisorpplate was washed 3 times with PBS+0.05% Tween-20. Cell lysate wastransferred directly to a washed Maxisorp plate and incubated overnightat 4° C. with gentle agitation. The plate was washed 6 times withPBS+0.05% Tween-20, then 100 μl anti-Phospho-tyrosine-HRP (DetectionAntibody 600 ng/ml) diluted in dilution buffer (PBS+0.5% BSA+0.05%Tween-20+5 mM EDTA) was added to each well and incubated for 2 hours atroom temperature. The plate was washed 6 times with PBS+0.05% Tween20and 100 μl Tetramethylbenzidine (TMB, Calbiochem) was added to each wellfor 20 minutes at room temperature. The reaction was stopped by additionof 50 μl 1 M H₂SO₄ and the absorbance was read at 450 nm (Thermo LabSystems plate reader).

The results are shown in FIG. 29. As illustrated, increasingconcentrations of U2-42.1 or U2-39.1 anti-HB-EGF antibody preparationsled to increased inhibition of HB-EGF-induced HER4 phosphorylation.

K. Example 11 Monoclonal Antibody Cross-Reactivity

This Example provides further data showing cross reactivity ofanti-HB-EGF antibody preparations for the cyno HB-EGF, mouse HB-EGF andthe related EGF-like growth factor Amphiregulin. Following cloning ofthe monkey and mouse form of HB-EGF, each expression construct wastransfected in HEK293 cells and anti-HB-EGF antibodies were tested ontheir ability to bind these proteins in a FACS experiment. Amphiregulincross reactivity was tested by ELISA format assay.

1. Cloning Cyno HB-EGF

In the present study, cyno HB-EGF plasmids were prepared. The cynoHB-EGF cDNA was cloned by polymerase chain reaction (PCR) from cynokidney cDNA with primers based on the sequence of cyno HB-EGF.

The primers used for the amplification of cyno HB-EGF were as follows:

Forward primer: (SEQ ID NO: 1078)5′-GGG TTA ACG CCA CCA TGA AGC TGC TGC CGT  CG-3′ Reverse primer:(SEQ ID NO: 1079) 5′-CCG CTC GAG GTG GGA ATT AGT CAT GCC C -3′

The PCR product was digested with Hpa1 and Xho1 and ligated intopcDNA3.1 digested with Hind3. After purification, cyno HB-EGF plasmidswere transformed into DH5α bacterial cells and multiplied underampicilin selection. The plasmid was then highly expressed in ampicilinselection media using a single transformed colony. After purifying usinga commercially available DNA-purification kit, cyno HB-EGF plasmids weretransiently transfected in HEK293T cells.

2. Cloning Mouse HB-EGF

In the present study, mouse HB-EGF plasmids were prepared. The mouseHB-EGF cDNA was cloned by polymerase chain reaction (PCR) from mouselung cDNA with primers based on the sequence of mouse HB-EGF.

The primers used for the amplification of mouse HB-EGF were as follows:

Forward primer: (SEQ ID NO: 1080)5′-GGA ATT CGC CAC CAT GAA GCT GCT GCC GTC  G-3′ Reverse primer:(SEQ ID NO: 1081) 5′-CCG CTC GAG GTG GGA GCT AGC AGC CAC GCC-3′

The PCR product and pcDNA3.1 vector DNA were digested with EcoR1 andXho1 and ligated. After purification, mouse HB-EGF plasmids weretransformed into DH5α bacterial cells and multiplied under ampicillinselection. The plasmid was then highly expressed in ampicillin selectionmedia using a single transformed colony. After purifying using acommercially available DNA-purification kit, mouse HB-EGF plasmids weretransiently transfected in HEK293T cells.

3. Transfection and Expression of Cyno and Mouse HB-EGF

To screen for cross-reactivity of antibodies provided herein HEK293Tcells were transiently transfected with either cyno or mouse HB-EGFplasmids using a Ca-phosphate method and subsequently analysed by FACSanalysis.

Therefore, 30 hours before transfection, 3×106 HEK293T-cells were seededin 16 ml on a 15 cm-cell culture plate and incubated at 7% CO₂ and 37°C. 32 μg DNA of either cyno or mouse HB-EGF DNA or empty vector in 720μl ddH₂O were mixed with 2.5 M CaCl₂ and 2×BBS (pH6.96) and incubated atroom temperature for 10 minutes. After incubation, the solution wasadded drop wise onto the cells and incubated at 3% CO₂ and 37° C. for 8hours. After soaking the media, the cells were incubated with freshgrowing media at 7% CO₂ and 37° C. for 24 hours.

4. FACS Analysis was Performed to Screen for Cross-Reactivity of theAntibodies

Therefore, 2×10⁵ transfected cells were harvested with 10 mM EDTA inPBS, resuspended in FACS-buffer (PBS, 3% FCS, 0.4% azide) and seeded ona 96-well round bottom plate. After centrifugation for 3 min at 1000 rpmto remove supernatant, the cells were resuspended in anti-HB-EGFantibody dilution (100 μl/well) and incubated at 4° C. for 45 min. Thecells were washed twice with FACS buffer and resuspended with secondaryantibody (100 μl/well) donkey-anti-human-PE (Jackson) diluted 1:100 inFACS buffer. The cell suspensions were incubated at 4° C. in the darkfor 30 min, washed twice with FACS buffer and analyzed (FACS, BeckmanCoulter).

FIG. 30A shows that three mAb preparations cross-react with pro-HB-EGFfrom Cynomolgus monkeys.

FIG. 30B shows that the U2-45 mAb preparation cross-reacts with mouseHB-EGF as detected by FACS analysis. Further testing showed thatantibodies U2-46 and U2-51 were also detecting mouse HB-EGF. However,the antibody U2-45.1 was only very weakly (5-10%) neutralizing mouseHB-EGF induced tyrosine phosphorylation of the EGFR.

5. Protocol for Amphiregulin Cross Reactivity ELISA Assay

Different concentrations of Amphiregulin (R & D systems, conc. 1 ng/ml,10 ng/ml, 100 ng/ml in PBS) were coated overnight at 4° C. in a 96-wellplate (Nunc, Maxisorp). Following plate wash (6-times) with washingbuffer (PBS+0.05% Tween 20; 150 μl per well), the plate was incubatedwith blocking buffer (PBS+0.5% BSA, 100 μl/well) for 4 hours at roomtemperature. The plate was washed 6-times with washing buffer. Asprimary antibody 5 μg/ml purified human anti-HB-EGF antibodies in Abdilution buffer (PBS containing 0.5% BSA, 0.05% Tween 20, 5 mM EDTA)were used and incubated for 90 minutes at room temperature. The platewas washed 6-times with washing buffer and a secondary anti human-POD(Dianova) antibody was added (1:10 000 in PBS, 0.5% BSA, 0.05% Tween 20,5 mM EDTA) and incubated for 60 minutes at room temperature.

The plate was washed 6-times with washing buffer and the TMB substrate(Merck Biosciences) was added for 15 minutes at room temperature. Afterstopping the development of the blue color by adding 100 μl of 250 mMHCl the absorbance was measured at 450 nm with a plate reader (Thermolabsystems).

FIG. 30C shows that antibody U2-45 and weakly antibody U2-46 bind toAmphiregulin as determined by an ELISA format assay. The U2-45 mAb bindsAmphiregulin but the U2-45.1 K_(D) for Amphiregulin was only 8 nM(versus 0.043 nM for HB-EGF). The U2-45.1 mAb was also non-neutralizingfor AR.

L. Example 12 Kinetic Exclusion Assay Analysis of K_(d) Values forAnti-HB-EGF Mabs U2-42.2, U2-39.1, U2-45.3, U2-26.2, and U2-34.1

The K_(D)s of mAbs U2-42.2, U2-39.1, U2-45.3, and U2-26.2, and U2-34.1binding to human HB-EGF, were determined using KinExA technology. Forthis purpose, a KinExA 3000 instrument was utilized. For all mAbtitrations, 50 mg of azlactone beads were coupled with HB-EGF (˜29 μg)in 50 mM sodium carbonate buffer, pH 9.0 overnight at 4° C. Afterconjugation of HB-EGF to the beads, the beads were centrifuged andwashed once with blocking buffer (1 M Tris buffer, pH 8.3, 10 mg/ml BSA)and centrifuged again, and then incubated in blocking buffer for one totwo hours at ˜23° C. in order to block any remaining reactive azlactonegroups present on the surface of the beads. After blocking, the beadswere transferred to a standard KinExA bead vial and placed on theinstrument.

MAb U2-42.2:

A dual curve analysis was performed to determine the K_(D). Twelvesolutions containing a nominal mAb binding site concentration of 37 pMwere titrated with increasing concentrations of HB-EGF for theK_(D)-controlled titration, and 1110 pM binding site was titrated in themAb-controlled titration curve. Each solution had a total volume of 10ml (K_(D)-controlled) or 2 ml (mAb-controlled) and was allowed toequilibrate for 30-36 hours (K_(D)-controlled) or 6 hours(mAb-controlled) at ˜23° C. All solutions for the titration wereprepared using volumetric glassware and the HB-EGF concentrations variedfrom 10.5 nM to 205 fM. The instrument method used for the analysis ofthese solutions consisted of a bead packing step in which the beads werepacked into a glass capillary, and the equilibrated solutions wereflowed through the bead column at 0.25 ml/min for 10 min (2.5 ml,K_(D)-controlled) or 1 min (0.25 ml, mAb-controlled) in triplicate.Subsequently, a fluorescently labeled cy-5 goat anti-human (Fc specific)polyclonal antibody at 3.4 nM (K_(D)-controlled) or 1.1 nM(mAb-controlled) was flowed through the bead pack for 2 min at 0.5ml/min to label the free mAb binding site captured on the beads. Thefluorescence emission from the bead pack was measured at 670 nm withexcitation at 620 nm.

The resulting fluorescence measurements were converted into %-free mAbbinding site versus total antigen concentration as standardly done withthe accompanying KinExA software package (version 1.0.3). The dualtitration curves were fit with the KinExA software to a 1:1 equilibriumisotherm with drift correction factors included.

The value of the K_(D) that fit the data optimally was 53 pM with lowand high 95% confidence limits at 34 pM and 81 pM, respectively.

MAb U2-39.1:

A K_(D)-controlled titration curve was performed to determine the K_(D).Twelve solutions containing a nominal mAb binding site concentration of55 pM were titrated with increasing concentrations of HB-EGF. Eachsolution had a total volume of 10 ml and was allowed to equilibrate for30-36 hours at ˜23° C. All solutions for the titration were preparedusing volumetric glassware and the HB-EGF concentrations varied from10.5 nM to 205 fM. The instrument method used for the analysis of thesesolutions consisted of a bead packing step in which the beads werepacked into a glass capillary, and the equilibrated solutions wereflowed through the bead column at 0.25 ml/min for 10 min (2.5 ml) intriplicate. Subsequently, a fluorescently labeled cy-5 goat anti-human(Fc specific) polyclonal antibody at 3.4 nM was flowed through the beadpack for 2 min at 0.5 ml/min to label the free mAb binding site capturedon the beads. The fluorescence emission from the bead pack was measuredat 670 nm with excitation at 620 nm. The resulting fluorescencemeasurements were converted into % free mAb binding site versus totalantigen concentration using the accompanying KinExA software package(version 1.0.3). Owing to ligand nonspecific binding to the bead pack,the titration curve was fit with the KinExA software to a 1:1equilibrium isotherm with a term for ligand nonspecific bindingincluded.

The value of the K_(D) that fit the data optimally was 7.8 pM with lowand high 95% confidence limits at 5.6 pM and 11 pM, respectively.

MAb U2-45.3:

A dual curve analysis was performed to determine the K_(D). Twelvesolutions containing a nominal mAb binding site concentration of 40 pMwere titrated with increasing concentrations of HB-EGF for theK_(D)-controlled titration, and 1060 pM binding was titrated in themAb-controlled titration curve. Each solution had a total volume of 10ml (K_(D)-controlled) or 2 ml (mAb-controlled) and was allowed toequilibrate for 30-36 hours (K_(D)-controlled) or 6 hours(mAb-controlled) at ˜23° C. All solutions for the titration wereprepared using volumetric glassware and the HB-EGF concentrations variedfrom 5.25 nM to 102 fM. The instrument method used for the analysis ofthese solutions consisted of a bead packing step in which the beads werepacked into a glass capillary, and the equilibrated solutions wereflowed through the bead column at 0.25 ml/min for 10 min (2.5 ml,K_(D)-controlled) or 1 min (0.25 ml, mAb-controlled) in triplicate.Subsequently, a fluorescently labeled cy-5 goat anti-human (Fc specific)polyclonal antibody at 3.4 nM (K_(D)-controlled) or 1.1 nM(mAb-controlled) was flowed through the bead pack for 2 min at 0.5ml/min to label the free mAb binding site captured on the beads. Thefluorescence emission from the bead pack was measured at 670 nm withexcitation at 620 nm. The resulting fluorescence measurements wereconverted into % free mAb binding site versus total antigenconcentration as standardly done with the accompanying KinExA softwarepackage (version 1.0.3). The dual titration curves were fit with theKinExA software to a 1:1 equilibrium isotherm with drift correctionfactors included.

The value of the K_(D) that fit the data optimally was 43 pM with lowand high 95% confidence limits at 27 pM and 65 pM, respectively.

MAb U2-26.2:

A dual curve analysis was performed to determine the K_(D). Twelvesolutions containing a nominal mAb binding site concentration of 41 pMwere titrated with increasing concentrations of HB-EGF for theK_(D)-controlled titration, and 1060 pM binding site was titrated in themAb-controlled titration curve. Each solution had a total volume of 10ml (K_(D)-controlled) or 2 ml (mAb-controlled) and was allowed toequilibrate for 30-36 hours (K_(D)-controlled) or 6 hours(mAb-controlled) at ˜23° C. All solutions for the titration wereprepared using volumetric glassware and the HB-EGF concentrations variedfrom 2.63 nM-51.4 fM (K_(D)-controlled) and 5.26 nM-103 fM(mAb-controlled). The instrument method used for the analysis of thesesolutions consisted of a bead packing step in which the beads werepacked into a glass capillary, and the equilibrated solutions wereflowed through the bead column at 0.25 ml/min for 10 min (2.5 ml,K_(D)-controlled) or 1 min (0.25 ml, mAb-controlled) in triplicate.Subsequently, a fluorescently labeled cy-5 goat anti-human (Fc specific)polyclonal antibody at 3.4 nM (K_(D)-controlled) or 1.1 nM(mAb-controlled) was flowed through the bead pack for 2 min at 0.5ml/min to label the free mAb binding site captured on the beads. Thefluorescence emission from the bead pack was measured at 670 nm withexcitation at 620 nm. The resulting fluorescence measurements wereconverted into % free mAb binding site versus total antigenconcentration as standardly done with the accompanying KinExA softwarepackage (version 1.0.3). The dual titration curves were fit with theKinExA software to a 1:1 equilibrium isotherm with drift correctionfactors included.

The value of the K_(D) that fit the data optimally was 61 pM with lowand high 95% confidence limits at 37 pM and 100 pM, respectively.

MAb U2-34.1:

A K_(D)-controlled titration curve was performed to determine the K_(D).Twelve solutions containing a nominal mAb binding site concentration of40 pM were titrated with increasing concentrations of HB-EGF. Eachsolution had a total volume of 10 ml and was allowed to equilibrate for30-36 hours at ˜23° C. All solutions for the titration were preparedusing volumetric glassware and the HB-EGF concentrations varied from5.26 nM-103 fM. The instrument method used for the analysis of thesesolutions consisted of a bead packing step in which the beads werepacked into a glass capillary, and the equilibrated solutions wereflowed through the bead column at 0.25 ml/min for 10 minutes (2.5 ml) intriplicate. Subsequently, a fluorescently labeled cy-5 goat anti-human(Fc specific) polyclonal antibody at 5.1 nM was flowed through the beadpack for 2 minutes at 0.5 ml/min to label the free mAb binding sitecaptured on the beads. The fluorescence emission from the bead pack wasmeasured at 670 nm with excitation at 620 nm. The resulting fluorescencemeasurements were converted into % free mAb binding site versus totalantigen concentration as standardly done with the accompanying KinExAsoftware package (version 1.0.3). Owing to ligand nonspecific binding tothe bead pack, the titration curve was fit with the KinExA software to a1:1 equilibrium isotherm with a term for ligand nonspecific bindingincluded.

The value of the K_(D) that fit the data optimally was 59 pM with lowand high 95% confidence limits at 32 pM and 87 pM, respectively.

M. Example 13 Determination of Antibody Affinity Scatchard Analysis

Affinity measurements of antibodies provided herein were performed byindirect FACS Scatchard analysis. To perform this analysis 2×10⁵ cellsof interest were harvested with 10 mM EDTA in PBS, resuspended inFACS-buffer (PBS, 3% FCS, 0.4% azide) and seeded on a 96-well roundbottom plate. After centrifugation for 3 min at 1000 rpm to removesupernatant, the cells were resuspended in anti-HB-EGF antibody dilution(100 μl/well) starting with 20 μg/ml, diluted in 1:2 dilution steps.Cell suspensions were incubated at 4° C. for 45 min, washed twice withFACS buffer and resuspended with secondary antibody (100 μl/well)donkey-anti-human-PE (Jackson) diluted 1:100 in FACS buffer. The cellsuspensions were incubated at 4° C. in the dark for 30 min, washed twicewith FACS buffer and analyzed (FACS, Beckman Coulter).

According to the FACS Scatchard analysis, the fluorescence mean wascalculated for each measurement. Background fluorescence of cellswithout HB-EGF antibodies was subtracted from each fluorescence mean.Scatchard plot with x-value=fluorescence mean and y-value=fluorescencemean/concentration of mAb (nM) was generated. The K_(D) was taken as theabsolute value of 1/m of linear equation.

TABLE 5 FACS Scatchard Determined Affinities Of Antibodies U2-42 AndU2-39 On Three Different Human Cancer Cell Lines Cell line AntibodyDLD-1 NCI-ADR MDA-MB231 U2-42 4.88 5.98 0.41 U2-39 9.05 7.63 6.21

N. Example 14 Kinetic Exclusion Assay Analysis of K_(d) Values forAnti-HB-EGF Mab U2-45.3 Binding to Amphiregulin

A K_(D)-controlled titration curve was performed to determine the K_(D).Twelve solutions containing a nominal mAb binding site concentration of4.4 nM were titrated with increasing concentrations of Amphiregulin.Each solution had a total volume of 10 ml and was allowed to equilibratefor 30-36 hours at ˜23° C. All solutions for the titration were preparedusing volumetric glassware and the Amphiregulin concentrations variedfrom 1.23 μM to 24 pM. The instrument method used for the analysis ofthese solutions consisted of a bead packing step in which the beads werepacked into a glass capillary, and the equilibrated solutions wereflowed through the bead column at 0.25 ml/min for 1 minute (0.25 ml) intriplicate. Subsequently, a fluorescently labeled cy-5 goat anti-human(Fc specific) polyclonal antibody at 684 pM was flowed through the beadpack for 2 minutes at 0.5 ml/min to label the free mAb binding sitecaptured on the beads. The fluorescence emission from the bead pack wasmeasured at 670 nm with excitation at 620 nm. The resulting fluorescencemeasurements were converted into % free mAb binding site versus totalantigen concentration as standardly done with the accompanying KinExAsoftware package (version 1.0.3). Owing to ligand nonspecific binding tothe bead pack, only one replicate out of the three collected at eachconcentration (the highest three 2-fold Amphiregulin concentrations wereexcluded from the analysis) could be analyzed and fit with the KinExAsoftware to a 1:1 equilibrium isotherm.

The value of the K_(D) that fit the data optimally was 5.0 nM with lowand high 95% confidence limits at 3.1 nM and 7.7 nM, respectively.

O. Example 15 Selection Criterion for Top Antibody Preparations

The following criteria were used to identify the top antibodypreparations: potency in inhibiting TMPS, potency in directly inhibitingHB-EGF as measured by observing the degree to which the antibodiesinhibited tyrosine phosphorylation of EGF receptor and HER4, theaffinity of the antibodies for HB-EGF, the cross-reactivity of theantibodies for other molecules and the characteristics of the epitopes.

P. Example 16 Anti-Hb-Egf Antibodies Inhibit HB-EGF Stimulation of HuvecCellular Proliferation and Tube Formation

This Example illustrates that while HB-EGF stimulates human vascularendothelial cell (HUVEC) proliferation, anti-HB-EGF antibodies providedherein inhibit basal HUVEC cell proliferation. Also, as shown by thisExample, anti-HB-EGF antibodies inhibit HUVEC tube formation, which isan in vitro model for neo-angiogenesis. Antibody preparations thatinhibit HUVEC proliferation and/or angiogenesis are useful not only fortreating cancer but also for treating non-cancerous conditions involvingundesired angiogenesis (e.g., diabetic retinopathy).

1. Procedures: Determination of HB-EGF Expression on HUVEC Cells by FlowCytometry

HB-EGF expression on human endothelial cells was determined by FACSanalysis. Therefore, 2×10⁵ cells of interest were harvested with 10 mMEDTA in PBS, resuspended in FACS-buffer (PBS, 3% FCS, 0.4% azide) andplated on a 96-well round bottom plate. After centrifugation for 3 minat 1000 rpm, supernatant was removed, the cells were resuspended inanti-HB-EGF antibody dilution (100 μl/well, 10 μg/ml anti-HB-EGFantibody) and incubated at 4° C. for 45 min. The cells were washed twicewith FACS buffer and resuspended with secondary antibody (100 μl/well)anti-human-PE (Jackson) diluted 1:100 in FACS buffer. The cellsuspensions were incubated at 4° C. in the dark for 30 min, washed twicewith FACS buffer and analyzed (FACS, Beckman Coulter).

To test for the effects of anti-HB-EGF antibodies on HUVECproliferation, approximately 5000 HUVEC cells were seeded into each of48 wells containing media with EGM-2, hydrocortisone, ascorbic acid,gentamycin-amphothericin and 2% FCS containing bFGF, VEGF, EGF and IGF-1(Cambrex). After incubating the cells overnight at 37 C, the cells werewashed twice with PBS containing 0.5% FCS. The cells were then starved 8h in EGM-2, 0.5% FCS without supplementation of growth factors. HB-EGFor anti-HB-EGF antibody preparations were added in 500 μl starvationmedia.

Cells were then cultured for an additional 60 hours, trypsinized andcounted.

To test for the effects of anti-HB-EGF antibodies on HUVEC tubeformation, 200 μl growth factor reduced matrigel (BD biosciences) wasplated on 48 wells. 250 μl HUVEC medium was added per 48 well(EBM-2+hydrocortisone+ascorbic acid gentamycin-amphothericin+0.25% FCSfrom Cambrex). Following preincubation for 20 min, 20,000 HUVEC cells in50 μl medium+0.25% FCS containing HB-EGF (10 ng/ml) or U2-42, U2-39 orU2-45 anti-HB-EGF antibodies (10 μg/ml) were added. Tube formation wasmonitored by obtaining photomicrographs of representative areas of theculture wells. For a quantitative analysis closed areas of HUVEC tubeswere counted.

2. Results

As shown by the FACS analysis in FIG. 30, HB-EGF is expressed on HUVECs.The results of the cellular proliferation tests are provided in FIGS.32A-B. As shown in FIG. 32A, HB-EGF stimulates HUVEC cellularproliferation by about 38%. However, upon addition of anti-HB-EGFantibody preparations U2-42, U2-39 or U2-45, such stimulation ofcellular proliferation is inhibited by about 8% to 14% (FIG. 31B). Inthis assay, the U2-39 anti-HB-EGF antibody preparation provided thehighest level of inhibition.

The results of the tube formation tests are provided in FIGS. 33A-M.Control assays, without anti-HB-EGF antibodies shown in FIGS. 34A-C,show that HUVEC cells join to form circular structures or “tubes.”However, upon addition of anti-HB-EGF antibody preparations U2-42, U2-39or U2-45, such tube formation is inhibited. A summary of the number oftubes observed is provided in FIG. 33M. As shown in FIG. 33M, the U2-42anti-HB-EGF antibody preparation provided the highest acceleration oftube regression, followed by the U2-39 anti-HB-EGF antibody preparation.

Q. Example 17 Anti-HB-EGF Antibodies Inhibit HB-EGF-Stimulated and BasalColony Formation

Soft agar assays were conducted in order to investigate the ability ofthe antibodies provided herein to inhibit anchorage independent cellgrowth. The soft agar colony formation assay is a standard in vitroassay to test for transformed cells, as only such transformed cells cangrow in soft agar.

To perform this assay, OVCAR-8, BM-1640 and NCI-H226 cells wereincubated with 10 ng/ml HB-EGF and with anti-HB-EGF antibodies or IgG2(SIGMA) as negative control, at 20 μg/ml in IMDM medium (Gibco) andresuspended in 0.2% Difco noble agar. The cell suspension was plated ona 0.4% agar-underlayer in quadruplicate in a 96-well plate and overlaidwith IMDM medium. Colonies were allowed to form for approximately 14days and were then stained with 40 μl MTT (Sigma, 1 mg/ml in PBS) for 4hours. Stimulation of HB-EGF and inhibitory effects of anti-HB-EGFantibodies were quantified by HTSBonit (LemnaTec) colony formationsoftware.

In another assay, 750 or 1000 cells (depending on SkOV-3 clone 71 or 74,FIGS. 34D and E) were preincubated with anti-HB-EGF antibodies or IgG2(SIGMA) as negative control, at 20 μg/ml in IMDM medium (Gibco) for 30min at 37° C. and resuspended in 0.4% Difco noble agar (or 0.2% forclone 74). The cell suspension was plated on a 0.75% agar-underlayer(0.4% for clone 74) in quadruplicate in a 96-well plate and overlaidwith IMDM medium. In a similar assay, 2000 BxPC-3 cells (FIG. 34F) werepreincubated with 20 μg/ml anti-HB-EGF antibodies or 20 μg/ml IgG2(SIGMA) as negative control, in IMDM medium (Gibco) containing 20% FCSfor 30 min at 37° C. Cells were resuspended in 0.4% Difco noble agar andthe cell suspension was plated on a 0.75% agar-underlayer inquadruplicate in a 96-well plate. The wells were overlaid with IMDMmedium. Both layers contained 20% FCS.

Colonies were allowed to form for approximately 14 days and were thenstained with 40 μl MTT (Sigma, 1 mg/ml in PBS) for 4 hours. Results areshown in FIGS. 34A-F, which illustrate that HB-EGF stimulated colonyformation (FIG. 34A-C) and basal colony formation (FIGS. 34D-F) issignificantly reduced by the anti-HB-EGF antibodies. As shown, forexample, in FIG. 34A, HB-EGF stimulated OVCAR-8 cells to form asignificantly larger mean colony size than control OVCER-8 cellscultured without HB-EGF. However, when OVCAR-8 cells were cultured withanti-HB-EGF U2-39 antibodies in the presence of HB-EGF, mean colony sizewas reduced to a size similar to that observed for control cells withoutHB-EGF treatment (FIG. 34A). Similar results were observed for BM1604cells (derived from prostate cancer tissue) (see, FIG. 34B). Anti-HB-EGFU2-45 and U2-42 antibodies also inhibited BM1604 colony formation.

Anti-HB-EGF antibodies also inhibit HB-EGF-stimulated colony formationof NCI-H226 lung carcinoma cells (FIG. 34C). As shown in FIG. 36C, whenNCI-H226 cells were cultured with anti-HB-EGF U2-39 antibodies in thepresence of HB-EGF, mean colony size was reduced to a size similar tothat observed for control cells without HB-EGF treatment.

The numbers of colonies, as well as the colony size, are reduced by thetreatment with the present anti-HB-EGF antibodies (FIGS. 34D-F). Thus,FIG. 34D illustrates that anti-HB-EGF antibodies reduce the number ofbasal colonies formed by SkOV-3 HB-EGF clone 71 cells (derived fromSkOV-3 ovarian cancer cells stably transfected with a proHB-EGFexpression construct). As shown, control SkOV-3 cells overexpressingHB-EGF formed large numbers of colonies. However, when SkOV-3 HB-EGF cl.71 cells were cultured with either anti-HB-EGF U2-42 or U2-39 antibodiesin the presence of HB-EGF, the number of colonies was dramaticallyreduced. Similarly, anti-HB-EGF antibodies inhibit colony formation ofSkOV-3 (clone 74) cells, derived from ovarian cancer tissue, and BxPC3cells, derived from pancreatic adenocarcinoma tissue (FIGS. 34E-F).

These data indicate that colony formation and tumors by a large varietyof cancer cell types can be inhibited by the present anti-HB-EGFantibodies, including the U2-42, U2-39 and U2-45 antibody preparationsprovided herein.

R. Example 18 Anti HB-EGF Antibodies Inhibit Tumor Growth In Vivo

FIG. 37 illustrates the mean volume of pancreatic BxPC3 tumors formed inxenograft experiments with SCID mice. As shown, established tumor growthwas significantly inhibited in the presence of antibody preparationsU2-42 and/or U2-39 when compared to the vehicle control. In FIG. 38A itis shown that anti-HB-EGF antibodies U2-42, U2-39 and U2-45 inhibit theestablished growth of EFO-27 HB-EGF clone 58 cells in vivo. The effectof tumor growth inhibition could be shown to be dose-dependent, with 25mg/kg as a highly effective treatment while lower doses such as 1 or 5mg/kg were less efficient (FIG. 38B).

S. Example 19 Anti-HB-EGF Antibodies in Combination Therapy with theAntiegfr Antibody Erbitux

FIG. 35 shows that single agent inhibition of EFO-27 HB-EGF cl. 58 cellswith anti-HB-EGF antibodies is moderate to strong. However, in adose-controlled combination of anti-HB-EGF and anti-EGFR antibodies theinhibition of colony formation is extremely effective. Moreover, in vivoxenograft growth is strongly inhibited by the anti-HB-EGF and anti-EGFRantibody combination leading to a complete regression of ovarian cancertumor growth (FIG. 38C).

T. Example 20 HB-EGF Expression on a Variety of Cancer Cell Types

HB-EGF expression on human cancer cell-lines was determined by FACSanalysis. To perform this analysis 2×10⁵ cells were harvested with 10 mMEDTA in PBS, resuspended in FACS-buffer (PBS, 3% FCS, 0.4% azide) andtransferred to a 96-well round bottom plate. After centrifugation for 3min at 1000 rpm to remove supernatant, the cells were resuspended inanti-HB-EGF antibody dilution (100 μl/well) and incubated at 4° C. for45 min. The cells were washed twice with FACS buffer and resuspendedwith secondary antibody (100 μl/well) donkey-anti-human-PE (Jackson)diluted 1:100 in FACS buffer. The cell suspensions were incubated at 4°C. in the dark for 30 minutes, washed twice with FACS buffer andanalyzed (FACS, Beckman Coulter).

The results of these assays are shown in TABLE 6, below.

TABLE 6 HB-EGF Expression in Cancer Cells Cell line Tissue ExpressionLevel MDA-MB231 Breast ++ NCI-ADR Breast +++ ZR75-1 Breast −/+ MKN-1Gastric + MKN-28 Gastric +++ PPC1 Prostate ++ PC3 Prostate ++ HT144Melanoma −/+ MelGerlach Melanoma +++ IGROV-1 Ovarian + ES2 Ovarian ++SkOV-3 Ovarian + SkOV-8 Ovarian + TOV21G Ovarian ++ OVCAR-8 Ovarian +++Calu-6 Lung + NCI-H460 Lung ++ MS-751 Cervix ++ SIHA Cervix + HelaS3Cervix + U266 Myeloma − SCABER Bladder ++ HCT-116 Colon ++ HCT-15Colon + SW620 Colon ++

U. Example 21 Anti-HB-EGF Antibodies for the Detection of HB-EGF inTissue and Body Fluids by Immunohistochemistry and ELISA

Anti-HB-EGF antibodies U2-42 and U2-39 were tested for their ability tostain HB-EGF expressed in human fixed samples. As shown in FIG. 39A bothantibodies show a prominent membrane and cytoplasmic staining of humankidney tubular cells while the control does not show any staining. Inaddition to the immunohistochemical detection of HB-EGF in patienttissue samples HB-EGF is released as a growth factor into various bodyfluids. Based on human anti-HB-EGF antibodies as coating reagents anELISA was established which detects HB-EGF in liquid samples down tolevels below 40 μg/ml (FIG. 39B).

V. Example 22 Canonical Classes of Antibodies

The genes encoding top antibodies were sequenced as described in thelast Example. This sequence data was used to assign the antibodies tocanonical classes.

Chothia et al. have described antibody structure in terms of “canonicalclasses” for the hypervariable regions of each immunoglobulin chain(Chothia, et al., 1987, J. Mol. Biol., 196(4): 901-17). The atomicstructures of the Fab and VL fragments of a variety of immunoglobulinswere analyzed to determine the relationship between their amino acidsequences and the three-dimensional structures of their antigen bindingsites. Chothia et al. found that there were relatively few residuesthat, through their packing, hydrogen bonding or the ability to assumeunusual phi, psi or omega conformations, were primarily responsible forthe main-chain conformations of the hypervariable regions. Theseresidues were found to occur at sites within the hypervariable regionsand in the conserved α-sheet framework. By examining sequences ofimmunoglobulins having unknown structure, Chothia, et al. show that manyimmunoglobulins have hypervariable regions that are similar in size toone of the known structures and additionally contained identicalresidues at the sites responsible for the observed conformation.

Their discovery indicated that these hypervariable regions haveconformations close to those in the known structures. For five of thehypervariable regions, the repertoire of conformations appeared to belimited to a relatively small number of discrete structural classes.These commonly occurring main-chain conformations of the hypervariableregions were termed “canonical structures.” Further work by Chothia, etal., 1989; Nature 342:877-83) and others (Martin et al., 1996; J. Mol.Biol. 263:800-15) confirmed that there is a small repertoire ofmain-chain conformations for at least five of the six hypervariableregions of antibodies.

The complementarity determining regions (CDRs) of each antibodypreparation were analyzed to determine their canonical class. As isknown, canonical classes have only been assigned for CDR1 and CDR2 ofthe antibody heavy chain, along with CDR1, CDR2 and CDR3 of the antibodylight chain. The TABLES below summarize the results of the analysis. TheCanonical Class data is in the form of HCDR1-HCDR2-LCDR1-LCDR2-LCDR3(H1-H2-L1-L2-L3), wherein “HCDR” refers to the heavy chain CDR and“LCDR” refers to the light chain CDR. Thus, for example, a canonicalclass of 1-3-2-1-1 refers to an antibody that has a HCDR1 that fallsinto canonical class 1, a HCDR2 that falls into canonical class 3, aLCDR1 that falls into canonical class 2, a LCDR2 that falls intocanonical class 1, and a LCDR3 that falls into canonical class 1.

Assignments were made to a particular canonical class where the aminoacids in the antibody match with the amino acids defined for eachcanonical class. The amino acids defined for each canonical class can befound, for example, in the articles by Chothia, et al. referred toabove. TABLE 7 and TABLE 8 report the canonical class assignments foreach of the HB-EGF antibodies. Where there was no matching canonicalclass, the canonical class assignment is marked with a letter s and anumber, such as “s18”, meaning the CDR is of size 18.

TABLE 7 Antibody (sorted) H1-H2-L1-L2-L3 H3length U2-18.1 3-1-2-1-1 17U2-13.1 1-3-4-1-1 14 U2-19.1 3-1-3-1-1 13 U2-38.1 3-1-2-1-1 23 U2-21.13-1-3-1-s9  8 U2-15.1 1-3-4-1-1 14 U2-16.1 1-2-8-1-1 11 U2-30.11-2-3-1-1 13 U2-42.1 1-3-2-1-s9  8 U2-36.1 3-s18-2-1-s10 13 U2-22.11-3-3-1-1 11 U2-56.1 3-1-2-1-1  8 U2-24.1 3-s16-3-1-s9 12 U2-24.2.13-s16-3-1-s9 12 U2-14.1 1-3-4-1-1 14 U2-1.1 1-3-4-1-1  5 U2-32.11-1-3-1-1 15 U2-40.1 1-3-2-1-3  8 U2-5.1 1-3-4-1-1  6 U2-8.1 3-1-4-1-117 U2-39.1 1-3-2-1-1  6 U2-3.1 3-1-4-1-1 16 U2-43.1 1-1-2-1-1 12 U2-34.11-3-2-1-1 16 U2-26.1 3-1-3-1-s9  9 U2-41.1 1-3-2-1-1 12 U2-45.11-3-2-1-1 19 U2-54.1 3-1-2-1-1 15 U2-57.1 3-1-2-1-1  8 U2-12.11-3-2-1-s9  9 U2-46.1 1-3-2-1-1 16 U2-48.2 1-1-2-1-1 12 U2-6.1.11-3-4-1-1 11 U2-6.1.2 1-3-2-1-1 16 U2-58.1 3-s16-2-1-1  8 U2-51.11-3-2-1-1 16 U2-65.2 3-1-2-1-1  8 U2-53.1 1-1-2-1-1 12 U2-61.1 1-3-2-1-1 9 U2-28.1 3-1-3-1-s9 12

TABLE 8 H1-H2-L1-L2-L3 Antibody (sorted) H3 Length U2-43.1 1-1-2-1-1 12U2-48.2 1-1-2-1-1 12 U2-53.1 1-1-2-1-1 12 U2-32.1 1-1-3-1-1 15 U2-30.11-2-3-1-1 13 U2-16.1 1-2-8-1-1 11 U2-39.1 1-3-2-1-1  6 U2-61.1 1-3-2-1-1 9 U2-41.1 1-3-2-1-1 12 U2-34.1 1-3-2-1-1 16 U2-46.1 1-3-2-1-1 16U2-6.1.2 1-3-2-1-1 16 U2-51.1 1-3-2-1-1 16 U2-45.1 1-3-2-1-1 19 U2-40.11-3-2-1-3  8 U2-42.1 1-3-2-1-s9  8 U2-12.1 1-3-2-1-s9  9 U2-22.11-3-3-1-1 11 U2-1.1 1-3-4-1-1  5 U2-5.1 1-3-4-1-1  6 U2-6.1.1 1-3-4-1-111 U2-13.1 1-3-4-1-1 14 U2-15.1 1-3-4-1-1 14 U2-14.1 1-3-4-1-1 14U2-56.1 3-1-2-1-1  8 U2-57.1 3-1-2-1-1  8 U2-65.2 3-1-2-1-1  8 U2-54.13-1-2-1-1 15 U2-18.1 3-1-2-1-1 17 U2-19.1 3-1-3-1-1 13 U2-21.13-1-3-1-s9  8 U2-26.1 3-1-3-1-s9  9 U2-28.1 3-1-3-1-s9 12 U2-313-1-4-1-1 16 U2-8.1 3-1-4-1-1 17 U2-58.1 3-s16-2-1-1  8 U2-24.13-s16-3-1-s9 12 U2-24.2.1 3-S16-3-1-s9 12 U2-36.1 3-s18-2-1-s10 13U2-38.1 3-1-2-1-1 23

TABLE 9 is an analysis of the number of antibodies per class. The numberof antibodies having the particular canonical class designated in theleft column is shown in the right column.

The most commonly seen structure is 1-3-2-1-1. Eight out of a total of40 mAbs had this combination.

TABLE 9 Number of Anti-HB-EGF Antibodies in Each Canonical ClassCombination H1-H2-L1-L2-L3 Count 1-1-2-1-1 3 1-1-3-1-1 1 1-2-3-1-1 11-2-8-1-1 1 1-3-2-1-1 8 1-3-2-1-3 1 1-3-2-1-s9 2 1-3-3-1-1 1 1-3-4-1-1 63-1-2-1-1 5 3-1-3-1-1 1 3-1-3-1-S9 3 3-1-4-1-1 2 3-s16-2-1-1 13-s16-3-1-s9 2 3-s18-2-1-s10 1 3-1-2-1-1 1

W. Example 23 Epitope Mapping of Anti HB-EGF Antibodies

This Example describes the mapping of epitopes recognized by antibodypreparations.

Antibodies tested: Five XenoMouse derived human monoclonal antibodypreparations capable of neutralizing the activity of HB-EGF wereanalyzed: U2-39; U2-42; U2-45; U2-26 and U2-19. Four of these monoclonalantibody preparations were shown to be specific for human HB-EGF whileantibody preparation U2-45 exhibited some cross-reactivity with mouseHB-EGF and human Amphiregulin. All of these neutralizing antibodypreparations map to MCAB Bins 7 & 8 (see, below).

1. Epitope Mapping

The human HB-EGF cDNA was isolated from HeLa mRNA by PCR amplificationand the mature HB-EGF sequence was cloned into a pSecTAg vector as amyc-His fusion protein, using the Ig kappa signal peptide sequence. Themature HB-EGF polypeptide was expressed in 293T cells, with secretioninto the media.

The diphtheria toxin binding site of HB-EGF was mutated as well as theEGF receptor binding site by site-directed mutagenesis.

The short form of HB-EGF (Loukianov et al; Gene 195:81-86), missing thethird disulphide bond of the EGF-like domain, was also cloned.

The EGF-like domain of HB-EGF having SEQ ID NO:1082 (DPCLRKYKDFCIHGECKYVKELRAPSCICHPGYHGERCHGLSLP) was cloned into pSecTag andexpressed and secreted as a Myc-His fusion protein.

2. Results

All of the U2-39; U2-42; U2-45; U2-26 and U2-19 antibody preparationsrecognize a discontinuous epitope. None of the antibodies recognize theShort form of the HB-EGF.

The binding site for all five antibody preparations is within theEGF-like domain, which included residues 44-86 of the mature proteinhaving the following sequence: DPCLRKYKDFCIHGECKYVKELRAPSCICHPGYHGERCHGLSLP (SEQ ID NO:1082). The third disulfide bond in the EGF-likedomain is required for binding of all of the U2-39; U2-42; U2-45; U2-26and U2-19 antibody preparations.

3. Structure-Function Analysis of Antibody Binding Site by Site DirectedMutagenesis

Twelve independent mutations were created in HB-EGF by replacing one tofour residues within the EGF-like domain of human pro-HB-EGF with thecorresponding amino acid residue normally found in the mouse pro-HB-EGF.

Given that many of the present antibody preparations were speciesselective, site-directed mutagenesis for the purpose of identifyingHB-EGF epitopes was done at known difference between the human HB-EGFprotein and related proteins from different species. In particular, theamino acid differences between human and mouse HB-EGF are as follows:K122R; V124L; K125Q; I133K; and H135L.

A complete list of HB-EGF mutant polypeptide sequences for epitopemapping is provided in TABLE 10. Mutant HB-EGF nucleic acids encodingthe desired mutant protein were transiently expressed in 293T cells, andmonoclonal antibody binding was measured by ELISA.

Thus, HB-EGF mutant polypeptides were made with these mutations and suchmutant polypeptides were tested to ascertain if the present antibodypreparations still bound to the HB-EGF mutant polypeptide. If not, thenthe mutated amino acid was likely important for antibody binding, andhence formed an important part of an epitope.

TABLE 11 summarizes the binding results obtained, where “Yes” indicatesthat binding took place despite the indicated mutation, “No” indicatesthat binding was substantially eliminated by the indicated mutation, and“Reduced” indicates that reduced binding occurred when the mutation waspresent.

TABLE 10 Wild  LGKKRDPCLRKYKDFCIHGE- SEQ ID typeCKYVKELRAPSCICHPGYHGERCHGLSLP NO: F115Y--------------Y----------------------------------- 1083 L127F---------------------------F---------------------- 1084 E141H-----------------------------------------H-------- 1085 K122R;----------------------R-LQ------------------------ 1086 V124L; K125QF115Y; --------------Y-------R-LQ------------------------ 1087 K122R;V124L; K125Q K122R; ----------------------R-LQ---------------H--------1088 V124L; K125Q; E141H I133K;---------------------------------K-L-------------- 1089 H135L F115Y;--------------Y------------------K-L-------------- 1090 I133K; H135LL127F; ---------------------------F-----K-L-------------- 1091 I133K;H135L L148T ------------------------------------------------T- 1092E141H; -----------------------------------------H------T- 1093 L148T117-120 ----------------LHDGV----------------------------- 1094 IHGEchanged to LHDGV

TABLE 11 Binding Results Construct/Mab U2-42.3 U2-39.1 U2-19.3 U2-26.1U2-45 1 Human proHB-EGF (WT) Yes Yes Yes Yes Yes 2 F115Y No No Yes YesReduced 3 L127F Yes No Yes Yes Yes 4 E141H No Yes Yes Yes Yes 5 K122R,V124L, K125Q Yes Yes Yes Yes Yes 6 F115Y, K122R, V124L, K125Q No No YesYes Reduced 7 K122R, V124L, K125Q, No Yes Yes Yes Yes E141H 8 I133K,H135L Yes Yes No No Yes 9 F115Y, I133K, H135L No No No No Reduced 10L127F, I133K, H135L, Yes No No No Yes 11 S147T Yes Yes Yes Yes Yes 12E141H, S147T No Yes Yes Yes Yes 13 IHGE (117-120) to LHDGV Yes Yes Ho NoNo Critical residues F115 & F115 & I133 or I133 or F115? E141 L127 H135H135

These binding studies indicate that the U2-42 and U2-39 antibodypreparations recognize the diphtheria toxin binding domain and the F115,L127 and E141 residues are important for diphtheria toxin binding.

Furthermore, when the F115Y or E141H mutations are present in HB-EGF,binding is substantially eliminated for the U2-42 antibody preparations.Thus, Phe-115 and Glu-141 are important for U2-42 antibody binding. TheU2-45 antibody preparation also appears to require Phe-115 becausebinding by this antibody preparation is reduced when Phe-115 is mutated.

The U2-39 antibody preparation requires Phe-115 and Leu-127 for bindingHB-EGF because mutation of either of those residues substantiallyeliminates antibody binding.

The U2-19, U2-26 and U2-45 antibody preparations bind the conservedregion between residues 117-120 (IHGE). As shown in TABLE 11, antibodypreparations U2-19 and U2-26 also recognize an epitope at Ile-133 and/orHis-135, because at least one of these residues is critical for theirbinding.

Based on their binding properties, of the antibodies were placed in therelationship “bins” listed in TABLE 12.

Binning is a method to group antibodies based on their competition forbinding to the antigen (see, Jia et al., 2004, J. Immunol. Methods288:91-98).

The assignment of bins depended on how different the observed bindingpatterns for all the antibodies tested are. Therefore, bins do notalways correlate with epitopes determined by other means and can be usedto only roughly define epitopes.

TABLE 12 Antibody Relationship Bins Bin#1 Bin#2 Bin#3 Bin#4 Bin#5 Bin#6Bin#7 Bin#8 U2-24.2 U2-1.3 U2-15.3 U2-13.3 U2-16.3 U2-18.3 U2-19.2U2-3.2 U2-32.3 U2-30.2 U2-14.3 U2-38.1 U2-21.3 U2-5.3 U2-2.1 1.19.2U2-34.1 U2-17.1 U2-57.1 U2-36.3 U2-26.2 U2-56.3 U2-58.3 U2-40.3 U2-41.3U2-61.1 U2-8.3 U2-45.3 U2-22.3 U2-46.3 U2- U2-39.1 48.2.1 U2-54.2 U2-6.1U2-51.2 U2-53.3 U2-28.2

In general the epitope mapping of U2-45 antibody preparation indicatesthat the Bin 7 antibody preparations cross-react with mouse HB-EGF andhuman Amphiregulin.

Mutations of F115 to Ala or Tyr affect the binding affinity of U2-45antibodies. However, U2-45 antibody binding was not affected bymutations of I133 and H135, which did affect some other Bin 7 antibodypreparations. When the IHGE human HB-EGF sequence was changed to LHDGV,which is present in mouse HB-EGF and human Aphiregulin, all Bin7antibody preparations failed to bind. Therefore, the IHGE residues(117-120) likely form the epitope for bin 7 antibody preparations.

Further site-directed mutagenesis studies involving binding of HB-EGFmutants to EGFR, indicate that residues including Asp-106 and Pro-107are both necessary for optimal binding of HB-EGF to the EGF receptor.Moreover, Leu-148, which is necessary for HB-EGF binding to the EGFreceptor, did not appear to be involved in the binding of any of theanti-HB-EGF antibody preparations.

X. Example 24 Sequences of Key Elements of Anti HB-EGF Antibodies

This Example provides the sequences of antibody preparations in FIGS.1-21.

Y. Example 25 Scratch Assay—Inhibition of HB-EGF-Induced Migration ofCLS354 Epithelial Equamous Carcinoma Cells (Mouth)

Scratch experiments were performed in order to investigate whether theantibodies of the invention block cell migration that would otherwise bedirectly induced by HB-EGF.

1×10⁶ CLS354 cells were seeded in medium (RPMI medium with 10% FCS) in 1ml on a 12-well plate and serum starved (medium with 0.5% FCS) overnight. After cells have reached a confluent layer, a scratch wasperformed in the middle of the well using a sterile plasic tip. Cellswere washed with PBS and scratched CLS354 cells were treated alone orcontaining 20 ng/ml HB-EGF in the presence or absence of 10 μg/ml U2-39,Erbitux or human IgG. The experiment was stopped after 12 hourincubation at 37° C. Medium was withdrawn, cells were washed with PBSand fixed with 100% ice-cold methanol at −20° C., stained withcrysal-violet, washed and dried over night. Photographes were taken fordocumentation.

FIG. 40A shows that HB-EGF treatment stimulates the closure of thescratch and that the antibody of invention, U2-39, inhibitsHB-EGF-mediated migration of CLS354 epithelial squamous carcinoma cellsinto the scratch.

Z. Example 26 Transmigration Assay—Inhibition of Hb-Egf-InducedMigration of Detroit 562 Epithelial Carcinoma Cells (Pharynx)

Transmigration experiments were performed in order to investigatewhether the antibodies of the invention block cell migration that wouldotherwise be directly induced by HB-EGF.

A 500 ml cell suspension of serum-starved human epithelial carcinomacells (50,000 cells) was placed in the top chamber of fibronectin-coatedtranswells (BD Falcon, 8 μm pores). Aliquots of 750 ml medium (Minimumessential medium (Eagle) in Earle's BSS with non-essential amino acids,sodium pyruvate (1 mM) and lactalbumin hydrolysate (0.1%), 90%; fetalbovine serum 10%, Pen.-Strept., 0.1% BSA) alone or containing 20 ng/mlHB-EGF (R&D Systems) in the presence or absence of 10 μg/ml human IgG,U2-39 or Erbitux antibodies were placed in the bottom chamber after 30min pre-incubation at 37° C. After incubation and migration for 6 hoursat 37° C., cells were fixed, stained with DAPI and transwells werephotographed for evaluation.

The result demonstrates that-HB-EGF antibody U2-39 effectively inhibitsHB-EGF-induced Detroit 562 epithelial carcinoma cell migrationcomparable to the inhibition of HB-EGF-mediated cell migration byErbitux treatment.

AA. Example 27 Spheroid-Based Cellular Angiogenesis Assay—Inhibition ofVegf-Stimulated Endothelial Cell Sprouting

Spheroid-based cellular angiogenesis assays were performed in order toinvestigate whether the antibodies of the invention are able to inhibitVEGF-induced endothelial cell (EC) sprouting in a collagen matrix.Primary human umbilical vein endothelial cells (HUVEC) were seeded outat 500 cells in a hanging drop on plastic dishes to allow overnightspheroid aggregation. 50 EC spheroids were seeded in 0.9 ml of collagensolution (2 mg/ml) and pipetted into individual wells of a 24 well plateto allow polymerization The antibody of invention U2-39 was directlymixed in the collagen solution before polymerization (differentconcentrations) and the growth factor VEGF-A (25 ng/ml) was added after30 min by pipetting 100 μl of a 10-fold concentrated working dilution ontop of the polymerized gel. Plates were incubated at 37° C. for 24 hoursand fixed by adding 4% paraformaldehyde. Sprouting intensity of ECspheroids was quantitated by an image analysis system determining thecumulative sprout length per spheroid using an inverted microscope andthe digital imaging software Analysis 3.2.

FIG. 41A depicts the mean of the cumulative sprout length of 10 randomlyselected spheroids per data point. FIG. 41B shows the relativeinhibition of the cumulative sprout length of 10 randomly selectedspheroids per data point by U2-39. The fitting of IC50 curves andcalculation of IC50 values was performed with GraphPad Prism 4.03.

The results of Example 26 demonstrate that the antibody of the inventionU2-39 inhibits VEGF-A-stimulated human umbilical vein endothelial cellsprouting in a dose-dependent manner in the spheroid-based assay using acollagen matrix. HUVEC sprouting was inhibited with an IC50 value of5.2×10⁻⁸ Molar.

AB. Example 28 Immunohistochemistry (Ihc) Analysis of Human TumorXenograft Samples—Inhibition of Cd31 Staining of Tumor In Vivo

In order to investigate the efficacy of the antibody of invention,U2-39, on inhibition of angiogenesis in vivo, human tumor xenograftstreated with U2-39 or Erbitux were analyzed by immunohistochemistryanalysis.

The human ovarian adenocarcinoma cell line EFO27 was geneticallyengineered to overexpress HB-EGF and the clone EFO27-CI58 was chosen forxenograft studies in SCID mice. 3×10⁶ EFO27-CI58 cells in 100 μlPBS/Matrigel (1:1) were injected subcutaneously into the left flank of 7week old female C.B-17 SCID mice. Tumor-bearing mice with mean tumorvolumes of 250 mm³ were randomized into groups containing 10 animals.Animals were treated intraperitoneally with weekly doses of 25 mg/kgU2-39 or 25 mg/kg Erbitux or control vehicle, PBS, for 3 weeks. After 28days mice were sacrificed, primary tumor tissues were collected and onehalf of the tumor was snap-frozen in liquid nitrogen and stored at −80°C.

5 to 8 μm sections of the tumor prepared on glass chamber slides werefixed in 100% acetone for 10 min at 4° C. and dried completely. To blockunspecific binding sites slides with fixed tumor sections were treatedwith Avidin D block (15 minutes), Biotin block (15 minutes) and a 1.25%BSA solution (1 hour). Between each treatment step slides were washedtwice with PBS. For immunohistochemical examination of the tumorvasculature the expression of the classical endothelial cell markerCD31, also known as PECAM-1 (Platelet Endothelial Cell AdhesionMolecule-1) was analyzed by treatment of the slides with 2 μg/mlanti-CD31 antibody (diluted in 1.25% BSA solution and incubated for 2 hat room temperature in a humidified chamber). Detection was performed byapplying a biotinylated goat ant-rat IgG antibody (30 min at roomtemperature) and Alexa 546 Streptavidin (15 min in the dark). PBSwashing steps were performed between each treatment step. Sections weremounted with VECTASHIELD mounting medium with DAPI in the dark,photographed (fluorescence microscope) for documentation and stored at4° C.

FIG. 42 demonstrates that human tumor xenografts treated with U2-39 showa reduced endothelial cell marker staining (CD31 staining) compared toErbitux-treated or control treated tumor xenografts. This resultdemonstrates the anti-angiogenic efficacy of the antibody of inventionin vivo.

AC. Example 29 In Vivo Ovarian Tumor Xenograft Model—CombinationTreatment of U2-39 with Cisplatin and Avastin

In order to evaluate the anti-tumor efficacy of the antibody ofinvention administered as a monotherapy or in combination with Cisplatinor Avastin, an ovarian cancer xenograft study was conducted.

The human ovarian adenocarcinoma cell line EFO27 was geneticallyengineered to overexpress HB-EGF. The clone EFO27-CI58 was chosen forxenograft studies in SCID mice. 3×10⁶ EFO27-CI58 cells in 100 μlPBS/Matrigel (1:1) were injected subcutaneously into the left flank of 7week old female C.B-17 SCID mice. Tumor-bearing mice with mean tumorvolumes between 75 and 175 mm³ were randomized into groups containing 10animals. Animals were treated intraperitoneally with weekly doses of 25mg/kg U2-39, 25 mg/kg Avastin or 5 mg/kg Cisplatin or control vehicle,PBS. Combination of U2-39 with Avastin was given at 12.5 mg/kg each andcombination of U2-39 with Cisplatin was given at 25 mg/kg antibody with5 mg/kg Cisplatin. Primary tumor sizes were determined 3 times a week.Following calliper measurement, tumor size was calculated according tothe formula W2×L/2 with L=length and W=the perpendicular width of thetumor. Kaplan-Meier log-rank method was used to define time toprogression to 500 mm³ (defined as “event” for statistical reasons).

FIG. 43A demonstrates that combination of U2-39 with Cisplatin led to astronger tumor reduction during the administration period than treatmentwith Cisplatin alone. In addition, a combination of U2-39 and Cisplatindelayed the time to progression of the median tumor size to 500 mm³compared to U2-39 monotherapy.

The result in FIG. 43B shows that combination of U2-39 with Avastinsignificantly delayed the time to progression to 500 mm³ tumor volumescompared to the treatment with Avastin as monotherapy although only halfof the single agent dose was administered.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and they are not necessarily restricted to the ordersof steps indicated herein or in the claims. As used herein and in theappended claims, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, a reference to “an antibody” includes a plurality (for example,a solution of antibodies or a series of antibody preparations) of suchantibodies, and so forth. Under no circumstances may the patent beinterpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein. Under no circumstances may thepatent be interpreted to be limited by any statement made by anyExaminer or any other official or employee of the Patent and TrademarkOffice unless such statement is specifically and without qualificationor reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and such modificationsand variations are considered to be within the scope of this inventionas defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

1. An isolated antigen binding protein that binds HB-EGF, comprising: A)one or more light chain complementary determining regions (CDRLs)selected from the group consisting of: (i) a CDRL1 selected from thegroup consisting of SEQ ID NOs:189-217; (ii) a CDRL2 selected from thegroup consisting of SEQ ID NOs:218-233; (iii) a CDRL3 selected from thegroup consisting of SEQ ID NOs:234-274; and (iv) a CDRL of (i), (ii) or(iii) that contains one or more amino acid substitutions, deletions orinsertions of no more than four amino acids; or B) one or more heavychain complementary determining regions (CDRHs) selected from the groupconsisting of: (i) a CDRH1 selected from the group consisting of SEQ IDNOs:275-299; (ii) a CDRH2 selected from the group consisting of SEQ IDNOs:300-331; (iii) a CDRH3 selected from the group consisting of SEQ IDNOs:332-372; and (iv) a CDRH of (i), (ii) or (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more thanfour amino acids.
 2. The isolated antigen binding protein of claim 1,comprising one or more light chain CDRLs of A), and one or more heavychain CDRHs of B).
 3. The isolated antigen binding protein of claim 1that comprises at least two CDRLs of A) and at least two CDRHs of B). 4.The isolated antigen binding protein of claim 1 that comprises saidCDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.
 5. The isolated antigenbinding protein of claim 1, wherein said CDRL of A) is selected from thegroup consisting of: (i) a CDRL1 selected from the group consisting ofSEQ ID NOs:189-217; (ii) a CDRL2 selected from the group consisting ofSEQ ID NOs:218-233; (iii) a CDRL3 selected from the group consisting ofSEQ ID NOs:234-274; and (iv) a CDRL of (i), (ii) or (iii) that containsone or more amino acid substitutions, deletions or insertions of no morethan two amino acids; said CDRH of B) is selected from the groupconsisting of: (i) a CDRH1 selected from the group consisting of SEQ IDNOs:275-299; (ii) a CDRH2 selected from the group consisting of SEQ IDNOs:300-331; (iii) a CDRH3 selected from the group consisting of SEQ IDNOs:332-372; and (iv) a CDRH of (i), (ii) or (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more thantwo amino acids; or C) one or more light chain CDRLs of A) and one ormore heavy chain CDRHs of B).
 6. The isolated antigen binding protein ofclaim 1, wherein said antigen binding protein comprises A) a CDRLselected from the group consisting of (i) a CDRL1 selected from thegroup consisting of SEQ ID NOs:189-217; (ii) a CDRL2 selected from thegroup consisting of SEQ ID NOs:218-233; and (iii) a CDRL3 selected fromthe group consisting of SEQ ID NOs:234-274; B) a CDRH selected from thegroup consisting of (i) a CDRH1 selected from the group consisting ofSEQ ID NOs:275-299; (ii) a CDRH2 selected from the group consisting ofSEQ ID NOs:300-331; and (iii) a CDRH3 selected from the group consistingof SEQ ID NOs:332-372; or C) one or more light chain CDRLs of A) and oneor more heavy chain CDRHs of B).
 7. The isolated antigen binding proteinof claim 6, wherein said antigen binding protein comprises A) a CDRL1 ofSEQ ID NOs:189-217, a CDRL2 of SEQ ID NOs:218-233, and a CDRL3 of SEQ IDNOs:234-274, and/or B) a CDRH1 of SEQ ID NOs:275-299, a CDRH2 of SEQ IDNOs:300-331, and a CDRH3 of SEQ ID NOs:332-372.
 8. The isolated antigenbinding protein of claim 1, wherein said antigen binding proteincomprises a light chain variable region (V_(L)) having at least 80%sequence identity with an amino acid sequence selected from the groupconsisting of SEQ ID NOs:94-141, and/or a heavy chain variable region(V_(H)) having at least 80% sequence identity with an amino acidsequence selected from the group consisting of SEQ ID NOs:142-186. 9.The isolated antigen binding protein of claim 8, wherein the V_(L) hasat least 90% sequence identity with an amino acid sequence selected fromthe group consisting of SEQ ID NOs:94-141, and/or the V_(H) has at least90% sequence identity with an amino acid sequence selected from thegroup consisting of SEQ ID NOs:142-186.
 10. The isolated antigen bindingprotein of claim 8, wherein the V_(L) is selected from the groupconsisting of SEQ ID NOs:94-141, and/or the V_(H) is selected from thegroup consisting of SEQ ID NOs:142-186.
 11. An isolated antigen bindingprotein that specifically recognizes at least an IHGE-containing epitopeand/or an EGF-like domain of HB-EGF.
 12. An isolated antigen bindingprotein that competes for binding with the antigen binding protein ofclaim
 1. 13. An isolated antigen binding protein that binds HB-EGF,wherein said antigen binding protein comprises: A) one or more lightchain CDRs (CDRLs) selected from the group consisting of: (i) a CDRL1with at least 80% sequence identity to SEQ ID NOs:189-217; (ii) a CDRL2with at least 80% sequence identity to SEQ ID NOs:218-233; and (iii) aCDRL3 with at least 80% sequence identity to SEQ ID NOs:234-274; B) oneor more heavy chain CDRs (CDRHs) selected from the group consisting of:(i) a CDRH1 with at least 80% sequence identity to SEQ ID NOs:275-299;(ii) a CDRH2 with at least 80% sequence identity to SEQ ID NOs:300-331;and (iii) a CDRH3 with at least 80% sequence identity to SEQ IDNOs:332-372; or C) one or more light chain CDRLs of A) and one or moreheavy chain CDRHs of B).
 14. The isolated antigen binding protein ofclaim 13, wherein said antigen binding protein comprises: A) one or moreCDRLs selected from the group consisting of: (i) a CDRL1 with at least90% sequence identity to SEQ ID NOs:189-217; (ii) a CDRL2 with at least90% sequence identity to SEQ ID NOs:218-233; and (iii) a CDRL3 with atleast 90% sequence identity to SEQ ID NOs:234-274; B) one or more CDRHsselected from the group consisting of: (i) a CDRH1 with at least 90%sequence identity to SEQ ID NOs:275-299; (ii) a CDRH2 with at least 90%sequence identity to SEQ ID NOs:300-331; and (iii) a CDRH3 with at least90% sequence identity to SEQ ID NOs:332-372; or C) one or more lightchain CDRLs of A) and one or more heavy chain CDRHs of B).
 15. Anisolated antigen binding protein that binds HB-EGF, the antigen bindingprotein comprising: A) a light chain complementary determining region(CDRL) selected from the group consisting of (i) a CDRL3 selected fromthe group consisting of SEQ ID NOs:234-274, (ii) a CDRL3 that differs inamino acid sequence from the CDRL3 of (i) by an amino acid addition,deletion or substitution of not more than two amino acids; and (iii) aCDRL3 amino acid sequence selected from the group consisting ofX₁QX₂X₃X₄X₅PX₆X₇, (SEQ ID NO: 1046)

 wherein X₁ is selected from the group consisting of I and M, X₂ isselected from the group consisting of A, G and S, X₃ is selected fromthe group consisting of I and T, X₄ is selected from the groupconsisting of H and Q, X₅ is selected from the group consisting of F, Land W, 7X₆ is selected from the group consisting of C, I, H, L and T, X₇is selected from the group consisting of S and T; QQX₁X₂X₃X₄X₅IT,(SEQ ID NO: 1047)

 wherein X₁ is selected from the group consisting of I and S, X₂ isselected from the group consisting of F and Y, X₃ is selected from thegroup consisting of F, I, S and Y, X₄ is selected from the groupconsisting of A, S and T, X₅ is selected from the group consisting of Pand S; X₁X₂X₃X₄X₅X₆X₇X₈T, (SEQ ID NO: 1048)

 wherein X₁ is selected from the group consisting of L and Q, X₂ isselected from the group consisting of K, N and Q, X₃ is selected fromthe group consisting of A, H, S and Y, X₄ is selected from the groupconsisting of H, N and Y, X₅ is selected from the group consisting of N,S and T, X₆ is selected from the group consisting of A, F, I, T, V andY, X₇ is selected from the group consisting of P and no amino acid, X₈is selected from the group consisting of F, L and P; QX₁X₂DX₃LPX₄X₅,(SEQ ID NO: 1049)

 wherein X₁ is selected from the group consisting of H and Q, X₂ isselected from the group consisting of C and Y, X₃ is selected from thegroup consisting of D, I, N, S and Y, X₄ is selected from the groupconsisting of F, I and L, X₅ is selected from the group consisting of A,S and T; QQX₁X₂X₃X₄PX₅X₆X₇, (SEQ ID NO: 1050)

 wherein X₁ is selected from the group consisting of H and Y, X₂ isselected from the group consisting of G and N, X₃ is selected from thegroup consisting of N and S, X₄ is selected from the group consisting ofS and W, X₅ is selected from the group consisting of P and no aminoacid, X₆ is selected from the group consisting of R and W, X₇ isselected from the group consisting of S and T; and X₁QYX₂X₃X₄X₅X₆X₇F,(SEQ ID NO: 1051)

 wherein X₁ is selected from the group consisting of H and Q, X₂ isselected from the group consisting of F and Y, X₃ is selected from thegroup consisting of G, I and S, X₄ is selected from the group consistingof F, I and T, X₅ is selected from the group consisting of M, P, S andT, X₆ is selected from the group consisting of F, L, R and W, X₇ isselected from the group consisting of S and T; and/or B) a heavy chaincomplementary determining region (CDRH) selected from the groupconsisting of (i) a CDRH3 selected from the group consisting of SEQ IDNOs:332-372, (ii) a CDRH3 that differs in amino acid sequence from theCDRH3 of (i) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (iii) a CDRH3 amino acid sequenceselected from the group consisting of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁DX₁₂,(SEQ ID NO: 1065)

 wherein X₁ is selected from the group consisting of E and S, X₂ isselected from the group consisting of D, G and no amino acid, X₃ isselected from the group consisting of D, N and no amino acid, X₄ isselected from the group consisting of G and no amino acid, X₅ isselected from the group consisting of G and no amino acid, X₆ isselected from the group consisting of W, Y and no amino acid, X₇ isselected from the group consisting of I, N and Y, X₈ is selected fromthe group consisting of A and Y, X₉ is selected from the groupconsisting of G, V and Y, X₁₀ is selected from the group consisting ofA, F and G, X₁₁ is selected from the group consisting of F, L and M, X₁₂is selected from the group consisting of V and Y; (SEQ ID NO: 1066)QX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁YX₁₂X₁₃X₁₄DX₁₅,

 wherein X₁ is selected from the group consisting of G and no aminoacid, X₂ is selected from the group consisting of K, L and Y, X₃ isselected from the group consisting of A, G and S, X₄ is selected fromthe group consisting of S, V and Y, X₅ is selected from the groupconsisting of A and G, X₆ is selected from the group consisting of G andno amino acid, X₇ is selected from the group consisting of T and noamino acid, X₈ is selected from the group consisting of S and no aminoacid, X₉ is selected from the group consisting of Y and no amino acid,X₁₀ is selected from the group consisting of W and Y, X₁₁ is selectedfrom the group consisting of G, S and Y, X₁₂ is selected from the groupconsisting of F and Y, X₁₃ is selected from the group consisting of Gand no amino acid, X₁₄ is selected from the group consisting of M and noamino acid, X₁₅ is selected from the group consisting of V and Y;(SEQ ID NO: 1067) X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄,

 wherein X₁ is selected from the group consisting of D, G, L, S and noamino acid, X₂ is selected from the group consisting of G, H, W, Y andno amino acid, X₃ is selected from the group consisting of A, F, W, Yand no amino acid, X₄ is selected from the group consisting of D, G, Q,T and no amino acid, X₅ is selected from the group consisting of G, I,Q, S and no amino acid, X₆ is selected from the group consisting of A,D, N, Q, S and no amino acid, X₇ is selected from the group consistingof G, Y and no amino acid, X₈ is selected from the group consisting ofD, Y and no amino acid, X₉ is selected from the group consisting of Yand no amino acid, X₁₀ is selected from the group consisting of A, E, Nand Y, X₁₁ is selected from the group consisting of G, P, T, V and Y,X₁₂ is selected from the group consisting of F and I, X₁₃ is selectedfrom the group consisting of D and Q, X₁₄ is selected from the groupconsisting of C, H, V and Y; (SEQ ID NO: 1068)X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇DX₁₈,

 wherein X₁ is selected from the group consisting of E, D and no aminoacid, X₂ is selected from the group consisting of G, R and no aminoacid, X₃ is selected from the group consisting of I, V, Y and no aminoacid, X₄ is selected from the group consisting of A, G, L and N, X₅ isselected from the group consisting of A, G, V and W, X₆ is selected fromthe group consisting of A, N, R and T, X₇ is selected from the groupconsisting of G, N, P and no amino acid, X₈ is selected from the groupconsisting of G, T and no amino acid, X₉ is selected from the groupconsisting of A and no amino acid, X₁₀ is selected from the groupconsisting of D, E and no amino acid, X₁₁ is selected from the groupconsisting of S, Y and no amino acid, X₁₂ is selected from the groupconsisting of G, Y and no amino acid, X₁₃ is selected from the groupconsisting of N, Y and no amino acid, X₁₄ is selected from the groupconsisting of Y and no amino acid, X₁₅ is selected from the groupconsisting of D, Y and no amino acid, X₁₆ is selected from the groupconsisting of A, G and no amino acid, X₁₇ is selected from the groupconsisting of F and M, X₁₈ is selected from the group consisting of I, Vand Y: (SEQ ID NO: 1069)X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁ X₂₂X₂₃,

 wherein X₁ is selected from the group consisting of A, D, G, S and T,X₂ is selected from the group consisting of A, E, G, L, N, R, Y and noamino acid, X₃ is selected from the group consisting of A, G, L, N, R,T, Y and no amino acid, X₄ is selected from the group consisting of D,G, R, S, V, Y and no amino acid, X₅ is selected from the groupconsisting of A, G, I, S, V, Y and no amino acid, X₆ is selected fromthe group consisting of F, G, L, R, V and no amino acid, X₇ is selectedfrom the group consisting of L, T, Y and no amino acid, X₈ is selectedfrom the group consisting of Y and no amino acid, X₉ is selected fromthe group consisting of Y and no amino acid, X₁₀ is selected from thegroup consisting of D and no amino acid, X₁₁ is selected from the groupconsisting of S and no amino acid, X₁₂ is selected from the groupconsisting of S and no amino acid, X₁₃ is selected from the groupconsisting of G and no amino acid, X₁₄ is selected from the groupconsisting of D, L, M, S, Y and no amino acid, X₁₅ is selected from thegroup consisting of H, I, P, V, W and no amino acid, X₁₆ is selectedfrom the group consisting of F, G, L, R, S, Y and no amino acid, X₁₇ isselected from the group consisting of D, F, V, W, Y and no amino acid,X₁₈ is selected from the group consisting of C, F, L, P, S and Y, X₁₉ isselected from the group consisting of D, F, G and Y, X₂₀ is selectedfrom the group consisting of A, C, G, P, R, V and Y, X₂₁ is selectedfrom the group consisting of F, L, M, S and no amino acid, X₂₂ isselected from the group consisting of A, D and no amino acid, X₂₃ isselected from the group consisting of I, L, V, Y and no amino acid;X₁YSSGWX₂X₃YGX₄X₅DX₆, (SEQ ID NO: 1070)

 wherein X₁ is selected from the group consisting of M and V, X₂ isselected from the group consisting of S and no amino acid, X₃ isselected from the group consisting of F and no amino acid, X₄ isselected from the group consisting of V and no amino acid, X₅ isselected from the group consisting of F and M, X₆ is selected from thegroup consisting of V and Y; and RX₁X₂X₃PFX₄Y, (SEQ ID NO: 1071)

 wherein X₁ is selected from the group consisting of G, H, L, N and R,X₂ is selected from the group consisting of E, T and W, X₃ is selectedfrom the group consisting of L, N, T and V, X₄ is selected from thegroup consisting of D and E.
 16. The isolated antigen binding protein ofclaim 15, said antigen binding protein further comprising: A) a CDRLselected from the group consisting of: (i) a CDRL1 selected from thegroup consisting of SEQ ID NOs:189-217; (ii) a CDRL1 that differs inamino acid sequence from the CDRL1 of (i) by an amino acid addition,deletion or substitution of not more than two amino acids; (iii) a CDRL1amino acid sequence selected from the group consisting ofX₁SSQSLX₂X₃SDGX₄TYLX₅, (SEQ ID NO: 1035)

 wherein X₁ is selected from the group consisting of K and R, X₂ isselected from the group consisting of L and V, X₃ is selected from thegroup consisting of H and Y, X₄ is selected from the group consisting ofK and N, X₅ is selected from the group consisting of N, S and Y;RASQX₁ISX₂YLN, (SEQ ID NO: 1036)

 wherein X₁ is selected from the group consisting of R, S and T, X₂ isselected from the group consisting of R and S; RASQX₁IX₂X₃X₄LX₅,(SEQ ID NO: 1037)

 wherein X₁ is selected from the group consisting of D, G, S and T, X₂is selected from the group consisting of A, R and S, X₃ is selected fromthe group consisting of H, I, N, R, S and T, X₄ is selected from thegroup consisting of D, W and Y, X₅ is selected from the group consistingof A, G and N; QASQDIX₁X₂X₃LN, (SEQ ID NO: 1038)

 wherein X₁ is selected from the group consisting of S and T, X₂ isselected from the group consisting of D and N, X₃ is selected from thegroup consisting of S and Y; RASQX₁VX₂X₃X₄X₅LA, (SEQ ID NO: 1039)

 wherein X₁ is selected from the group consisting of S and T, X₂ isselected from the group consisting of I and S, X₃ is selected from thegroup consisting of R and S, X₄ is selected from the group consisting ofS, N and no amino acid, X₅ is selected from the group consisting of Yand no amino acid; and KSSQX₁X₂LX₃X₄SNNKNYLX₅, (SEQ ID NO: 1040)

 wherein X₁ is selected from the group consisting of N and S, X₂ isselected from the group consisting of I and V, X₃ is selected from thegroup consisting of D and Y, X₄ is selected from the group consisting ofN, R and S, X₅ is selected from the group consisting of A and V; (iv) aCDRL2 selected from the group consisting of SEQ ID NOs:218-233; (v) aCDRL2 that differs in amino acid sequence from the CDRL2 of (iv) by anamino acid addition, deletion or substitution of not more than two aminoacids; and (vi) a CDRL2 amino acid sequence selected from the groupconsisting of X₁X₂SNX₃X₄S, (SEQ ID NO: 1041)

 wherein X₁ is selected from the group consisting of E and K, X₂ isselected from the group consisting of I and V, X₃ is selected from thegroup consisting of R and W, X₄ is selected from the group consisting ofD and F; X₁X₂SX₃LQS, (SEQ ID NO: 1042)

 wherein X₁ is selected from the group consisting of A and T, X₂ isselected from the group consisting of A, E and V, X₃ is selected fromthe group consisting of S and T; X₁ASX₂LQS, (SEQ ID NO: 1043)

 wherein X₁ is selected from the group consisting of A and V, X₂ isselected from the group consisting of S and T; DASX₁LET,(SEQ ID NO: 1044)

 wherein X₁ is selected from the group consisting of I and N; GASSRAT;(SEQ ID NO: 223) and WASX₁RES, (SEQ ID NO: 1045)

 wherein X₁ is selected from the group consisting of A and T; or B) aCDRH selected from the group consisting of: (i) a CDRH1 selected fromthe group consisting of SEQ ID NOs:275-299; (ii) a CDRH1 that differs inamino acid sequence from the CDRH1 of (i) by an amino acid addition,deletion or substitution of not more than two amino acids; (iii) a CDRH1amino acid sequence selected from the group consisting ofGYTX₁TX₂X₃X₄X₅X₆, (SEQ ID NO: 1052)

 wherein X₁ is selected from the group consisting of F and L, X₂ isselected from the group consisting of E, G and S, X₃ is selected fromthe group consisting of H, L and Y, X₄ is selected from the groupconsisting of G, S and Y, X₅ is selected from the group consisting of Iand M, X₆ is selected from the group consisting of H and S; GYX₁FTSYWIG,(SEQ ID NO: 1053)

 wherein X₁ is selected from the group consisting of R and S;GFTFX₁SX₂X₃MH, (SEQ ID NO: 1054)

 wherein X₁ is selected from the group consisting of R and S, X₂ isselected from the group consisting of H and Y, X₃ is selected from thegroup consisting of D and G; GFX₁FSX₂YX₃MX₄, (SEQ ID NO: 1055)

 wherein X₁ is selected from the group consisting of P and T, X₂ isselected from the group consisting of A, R and S, X₃ is selected fromthe group consisting of A and S, X₄ is selected from the groupconsisting of N and S; GX₁SX₂SX₃X₄X₅X₆X₇WX₈, (SEQ ID NO: 1056)

 wherein X₁ is selected from the group consisting of D and G, X₂ isselected from the group consisting of F, I and V, X₃ is selected fromthe group consisting of R, S and no amino acid, X₄ is selected from thegroup consisting of G, Y and no amino acid, X₅ is selected from thegroup consisting of D, G, S and no amino acid, X₆ is selected from thegroup consisting of A, S and Y, X₇ is selected from the group consistingof A and Y, X₈ is selected from the group consisting of N and S;GFSLSNARMGVS; (SEQ ID NO: 279) and GFSLX₁TGGVGVG, (SEQ ID NO: 1057)

 wherein X₁ is selected from the group consisting of S and N; (iv) aCDRH2 selected from the group consisting of SEQ ID NOs:300-331; (v) aCDRH2 that differs in amino acid sequence from the CDRH2 of (iv) by anamino acid addition, deletion or substitution of not more than two aminoacids; and (vi) a CDRH2 amino acid sequence selected from the groupconsisting of (SEQ ID NO: 1058) X₁X₂X₃X₄X₅X₆GX₇TX₈X₉X₁₀QKX₁₁X₁₂,

 wherein X₁ is selected from the group consisting of S and W, X₂ isselected from the group consisting of F and I, X₃ is selected from thegroup consisting of D, N and S, X₄ is selected from the group consistingof A and P, X₅ is selected from the group consisting of E, N and S, X₆is selected from the group consisting of D, N and S, X₇ is selected fromthe group consisting of E, G and N, X₈ is selected from the groupconsisting of I and N, X₉ is selected from the group consisting of C, Hand Y, X₁₀ is selected from the group consisting of A and T, X₁₁ isselected from the group consisting of F and L, X₁₂ is selected from thegroup consisting of D and G; IIYPX₁DSDX₂RYSPSFQG, (SEQ ID NO: 1059)

 wherein X₁ is selected from the group consisting of D and G, X₂ isselected from the group consisting of A, I and T;X₁IX₂X₃DGSX₄X₅X₆YX₇DSVX₈G, (SEQ ID NO: 1060)

 wherein X₁ is selected from the group consisting of F and V, X₂ isselected from the group consisting of S and W, X₃ is selected from thegroup consisting of D, S and Y, X₄ is selected from the group consistingof I, N and T, X₅ is selected from the group consisting of K and Q, X₆is selected from the group consisting of N, R and Y, X₇ is selected fromthe group consisting of A, T and V, X₈ is selected from the groupconsisting of K and R; X₁ISX₂SX₃X₄X₅X₆YYADSVKG, (SEQ ID NO: 1061)

 wherein X₁ is selected from the group consisting of A, H and Y, X₂ isselected from the group consisting of G, R and S, X₃ is selected fromthe group consisting of G and S, X₄ is selected from the groupconsisting of G, R and S, X₅ is selected from the group consisting of S,T and Y, X₆ is selected from the group consisting of I and T;(SEQ ID NO: 1062) X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁YX₁₂X₁₃SX₁₄KS,

 wherein X₁ is selected from the group consisting of E, R and Y, X₂ isselected from the group consisting of I and T, X₃ is selected from thegroup consisting of H, N and Y, X₄ is selected from the group consistingof C, H, S, T and Y, X₅ is selected from the group consisting of S andR, X₆ is selected from the group consisting of G and S, X₇ is selectedfrom the group consisting of G, K, S and T, X₈ is selected from thegroup consisting of T and W, X₉ is selected from the group consisting ofN and Y, X₁₀ is selected from the group consisting of N and no aminoacid, X₁₁ is selected from the group consisting of D and no amino acid,X₁₂ is selected from the group consisting of A and N, X₁₃ is selectedfrom the group consisting of P and V, X₁₄ is selected from the groupconsisting of L and V; X₁IFSNDEKSYSTSLKS, (SEQ ID NO: 1063)

 wherein X₁ is selected from the group consisting of H and L1; andLIYWNX₁X₂KRYSPSLX₃S, (SEQ ID NO: 1064)

 wherein X₁ is selected from the group consisting of D and V, X₂ isselected from the group consisting of D and E, X₃ is selected from thegroup consisting of K and R.
 17. The isolated antigen binding protein ofclaim 16, wherein said antigen binding protein comprises said firstamino acid sequence and said second amino acid sequence.
 18. Theisolated antigen binding protein of claim 17, wherein said first aminoacid sequence is covalently bonded to said second amino acid sequence.19. The isolated antigen binding protein of claim 17, wherein said firstamino acid sequence comprises said CDRL3 of SEQ ID NOs:234-274, CDRL2 ofSEQ ID NOs:218-233, and CDRL1 of SEQ ID NOs: 189-217, and said secondamino acid sequence comprises said CDRH3 of SEQ ID NOs:332-372, CDRH2 ofSEQ ID NOs:300-331, and CDRH1 of SEQ ID NOs:275-299.
 20. The isolatedantigen binding protein of any of claim 1-19, wherein said antigenbinding protein is a monoclonal antibody, a polyclonal antibody, arecombinant antibody, a human antibody, a humanized antibody, a chimericantibody, a multispecific antibody, or an antibody fragment thereof. 21.The isolated antigen binding protein of claim 20, wherein said antibodyfragment is a Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment, a Fvfragment, a diabody, or a single chain antibody molecule.
 22. Theisolated antigen binding protein of claim 20, wherein said antigenbinding protein is a human antibody.
 23. The isolated antigen bindingprotein of claim 20, wherein said antigen binding protein is amonoclonal antibody.
 24. The isolated antigen binding protein of any ofclaims 1-19 wherein said antigen binding protein is of the IgG1-,IgG2-IgG3- or IgG4-type.
 25. The isolated antigen binding protein ofclaim 24, wherein said antigen binding protein is of the IgG2- orIgG4-type.
 26. The isolated antigen binding protein of any of claims1-19, wherein said antigen binding protein is coupled to a labelinggroup.
 27. The isolate antigen binding protein of claim 26, wherein thelabeling group is a radioisotope, radionuclide, a fluorescent group, anenzymatic group, a chemiluminescent group, a biotinyl group, or apredetermined polypeptide group.
 28. The isolated antigen bindingprotein of any of claims 1-19, wherein said antigen binding protein iscoupled to an effector group.
 29. The isolated antigen binding proteinof claim 28, wherein said effector group is a radioisotope, aradionuclide, a toxin, a therapeutic group, or a chemotherapeutic group.30. The isolated antigen binding protein of claim 29, wherein saidtherapeutic group or chemotherapeutic group is calicheamicin,auristatin-PE, geldanamycin, maytanasine, or derivatives thereof.
 31. Anisolated antigen binding protein that competes for binding to humanHB-EGF with an antigen binding protein of one of claims 1-19.
 32. Theisolated antigen binding protein of claim 31, wherein said antigenbinding protein is a monoclonal antibody, a polyclonal antibody, arecombinant antibody, a human antibody, a humanized antibody, a chimericantibody, a multispecific antibody, or an antibody fragment thereof. 33.The isolated antigen binding protein of claim 32, wherein said antibodyfragment is a Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment, a Fvfragment, a diabody, or a single chain antibody molecule.
 34. Theisolated antigen binding protein of claim 32, wherein said antigenbinding protein is a human antibody.
 35. The isolated antigen bindingprotein of claim 32, wherein said antigen binding protein is amonoclonal antibody.
 36. The isolated antigen binding protein of any ofclaim 31, wherein said antigen binding protein is of the IgG1-,IgG2-IgG3- or IgG4-type.
 37. The isolated antigen binding protein ofclaim 36, wherein said antigen binding protein is of the IgG2- or theIgG4-type.
 38. The isolated antigen binding protein of any of claim 31,wherein said antigen binding protein is coupled to a labeling group. 39.The isolate antigen binding protein of claim 38, wherein the labelinggroup is a radioisotope, radionuclide, a fluorescent group, an enzymaticgroup, a chemiluminescent group, a biotinyl group, or a predeterminedpolypeptide group.
 40. The isolated antigen binding protein of claim 31,wherein said antigen binding protein is coupled to an effector group.41. The isolated antigen binding protein of claim 40, wherein saideffector group is a radioisotope, a radionuclide, a toxin, a therapeuticgroup, or a chemotherapeutic group.
 42. The isolated antigen bindingprotein of claim 41, wherein said therapeutic group or chemotherapeuticgroup is calicheamicin, auristatin-PE, geldanamycin, maytanasine, orderivatives thereof.
 43. The isolated antigen binding protein of one ofclaims 1-19, wherein said antigen binding protein reduces at leastpartially HB-EGF-mediated signal transduction.
 44. A nucleic acidmolecule encoding the antigen binding protein according to any one ofclaims 1-19.
 45. The nucleic acid molecule according to claim 44,wherein said nucleic acid molecule is operably linked to a controlsequence.
 46. A vector comprising a nucleic acid molecule according toclaim
 44. 47. A vector comprising a nucleic acid molecule according toclaim
 45. 48. A host cell comprising the nucleic acid molecule accordingto claim
 45. 49. A host cell comprising the vector according to one ofclaim 46 or
 47. 50. A method of making the antigen binding proteinaccording to any one of claims 1-19, comprising the step of preparingsaid antigen binding protein from a host cell that secretes said antigenbinding protein.
 51. A pharmaceutical composition comprising at leastone antigen binding protein according to any one of claims 1-19, andpharmaceutically acceptable carrier, diluents and/or adjuvants.
 52. Thepharmaceutical composition of claim 51, further comprises an additionalactive agent.
 53. The pharmaceutical composition according to claim 52,wherein the at least one further active agent is an anti-neoplasticagent.
 54. The pharmaceutical composition of claim 53, wherein theanti-neoplastic agent is an anti-tumor antibody.
 55. The pharmaceuticalcomposition of claim 54, wherein the anti-tumor antibody is an antibodydirected against a receptor tyrosine kinase.
 56. The pharmaceuticalcomposition of claim 53, wherein the anti-tumor antibody is directedagainst EGFR.
 57. The pharmaceutical composition of claim 51, for thediagnosis, prevention or treatment of a hyperproliferative disease. 58.The pharmaceutical composition to claim 57, wherein saidhyperproliferative disease is associated with HB-EGF expression.
 59. Thepharmaceutical composition according to claim 57, wherein saidhyperproliferative disease is associated with or accompanied by adisturbed, e.g., pathologically enhanced growth factor receptoractivation.
 60. The pharmaceutical composition of claim 59, wherein saidpathologically enhanced growth factor receptor activation is associatedwith or caused by a pathological increase in the activity of a G proteinand/or a G protein coupled receptor.
 61. The pharmaceutical compositionof claim 51 for the diagnosis, prevention or treatment of cancer. 62.The pharmaceutical composition claim 61, wherein said cancer is selectedfrom the group consisting of breast cancer, gastrointestinal cancer,pancreas cancer, prostate cancer, ovarian cancer, stomach cancer,endometrial cancer, salivary gland cancer, lung cancer, kidney cancer,colon cancer, colorectal cancer, thyroid cancer, bladder cancer, glioma,melanoma, carcinoma, in particular epithelial or squamous carcinoma,other HB-EGF expressing or overexpressing cancers, and formation oftumor metastases.
 63. Use of at least one antigen binding protein ofclaims 1-19, for the manufacture of a pharmaceutical composition for thediagnosis, prevention or treatment of a hyperproliferative disease. 64.The use according to claim 63, wherein said hyperproliferative diseaseis a hyperproliferative disease as defined in claim
 58. 65. A method fordiagnosing a condition associated with the expression of HB-EGF,comprising contacting a sample with an antigen binding protein of claims1-19, and determining the presence of HB-EGF in said sample.
 66. Themethod according to claim 65, wherein the condition is ahyperproliferative disease as defined in claim
 58. 67. A method forpreventing or treating a condition associated with the expression ofHB-EGF in a patient, comprising administering to a patient in needthereof an effective amount of at least one antigen binding protein ofclaims 1-19.
 68. The method according to claim 62, wherein the conditionis a hyperproliferative disease as defined in any one of claims 57-60.69. The method of claim 57, wherein the patient is a mammalian patient.70. A kit comprising a antigen binding protein of claims 1-19, a nucleicacid molecule of claim 44 or 45 or a vector according to claim 46 or 47.71. The kit of claim 70 comprising at least one further active agent.72. The kit of claim 71, wherein the further active agent is ananti-neoplastic agent.
 73. The pharmaceutical composition according toclaim 53, wherein the anti-neoplastic agent id Cisplatin or Avastin. 74.The pharmaceutical composition according to any of the claims 51 to 62which is to be administered as a monotherapy or in combination with afurther pharmaceutical composition preferably comprising ananti-neoplastic agent such as cisplatin or Avastin.